Mutant fragments of ospa and methods and uses relating thereto

ABSTRACT

The present invention relates to a polypeptide comprising a mutant fragment of an outer surface protein A (OspA), a nucleic acid coding the same, a pharmaceutical composition (particularly for use as a medicament of in a method of treating or preventing a  Borrelia  infection) comprising the polypeptide and/or the nucleic acid, a method of treating or preventing a  Borrelia  infection and a method of immunizing a subject.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/623,681, filed Feb. 17, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/802,991, filed Mar. 14, 2013 and now issued asU.S. Pat. No. 8,986,704, which claims the benefit under 35 U.S.C.§119(e) of U.S. provisional application Ser. No. 61/668,627, filed Jul.6, 2012, the disclosures of which are incorporated by reference hereinin their entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for theprevention and treatment of Borrelia infection.

BACKGROUND OF THE INVENTION

Lyme borreliosis, or Lyme disease, is the most commonly reportedtick-borne disease in Europe and North America. The disease is caused bythe arthropod-borne gram-negative-like spirochete, Borrelia burgdorferisensu lato (B. burgdorferi s.l.), and is an infection that can involvemultiple organs or tissues, resulting in skin, cardiac, musculoskeletaland neurological disorders. In most countries, Lyme borreliosis is not anotifiable disease and no exact data regarding annual incident rates areavailable. In the United States, the causative agent is B. burgdorferisensu stricto (B. burgdorferi s.s.) and Lyme borreliosis is localized tonorth-eastern, mid-Atlantic and upper north-central states. In 2010, atotal of about 30,000 cases of Lyme borreliosis were reported for the USto the Centers for Disease Control and Prevention (CDC). In Europe, B.afzelii and B. garinii are the main causative agents of Lymeborreliosis, as well as B. burgdorferi s.s. and B. bavariensis, whichcontribute to a lesser extent depending on the geographic location. Theprevalence of Lyme borreliosis varies considerably in different Europeancountries with an overall increased prevalence from west to east. Inmuch of Europe, the number of reported cases of Lyme borreliosis hasincreased since the early 1990s (e.g., the Czech Republic, Estonia,Lithuania; see Lyme borreliosis in Europe, WHO report of 2006), and thegeographic distribution of cases has also expanded.

In some risk groups, such as farmers, forestry workers, hikers, runnersor vacationers, seroprevalence and disease incidence rates haveincreased, as in children under 15 years of age and adults between 39and 59, without gender preference. This increased incidence of Lymeborreliosis is linked to changes in forest habitats as well as socialfactors. Environmental changes, such as forest fragmentation, have ledto a sharp reduction of rodent predators such as foxes and birds ofprey, which in turn has led to an increase in the mouse population, witha subsequent increase in the tick population. More recently, patchyreforestation has increased the number of deer and thus the number ofticks. Suburban sprawl and the increasing use of woodland areas forrecreation such as camping and hiking has brought humans into greatercontact with the larger number of tick Borrelia vectors. All of thesefactors together have contributed to a wider distribution of Borreliaand a higher incidence of Lyme borreliosis.

Antimicrobial agents are the principle method of treatment of Borreliainfection. The antibiotic used depends on the stage of the disease,symptoms, and the patient's allergies to medication. The length of theantibiotic course also depends on the stage of the disease and theseverity of symptoms. Early Lyme borreliosis is typically treated withoral tetracyclines, such as doxycycline, and semi-synthetic penicillins,such as amoxicillin or penicillin V. Arthritic and neurologicaldisorders are treated with high-dose intravenous penicillin G orceftriaxone. Up to 30% of Lyme borreliosis patients do not display theearly characteristic symptoms of infection with Borrelia, makingdiagnosis and treatment problematic. The antibiotic course can be long(up to several months) and sometimes ineffective and is thus debated inthe Borrelia field, especially during later-stage disease. Even in thecase of effective treatment of Borrelia, patients can be left withdebilitating fatigue, pain, or neurological symptoms for yearsafterwards referred to as post-treatment Lyme disease syndrome. Ingeneral, the use of antibiotics can have undesirable consequences, suchas the development of resistance by the target micro-organisms. Finally,antibiotic therapy may effectively cure Lyme borreliosis, but providesno protection against subsequent infections.

A monovalent OspA-based vaccine (LYMErix™) was approved and marketed inthe USA for the prevention of Lyme disease. However, heterogeneity inOspA sequences across different serotypes in Europe and elsewhereprecludes efficient protection with a vaccine based on OspA from asingle serotype.

Chimeric OspA molecules comprising the proximal portion from one OspAserotype, together with the distal portion form another OspA serotype,while retaining antigenic properties of both of the parent polypeptides,may be used in the prevention and treatment of Lyme disease orborreliosis (WO2011/143617, WO2011/143623).

X-ray crystallography and NMR analysis have been used to identifyimmunologically important hypervariable domains in OspA and have mappedthe LA-2 epitope to amino acids 203-257 (Ding et al., Mol. Biol. 302:1153-64, 2000).

Currently, there is no preventative medicament for Lyme borreliosis onthe market and thus there is a need in the art for the development ofsuch a medicament that can provide effective protection against avariety of species of Borrelia that are present in the USA, Europe andelsewhere.

SUMMARY OF THE INVENTION

The present invention relates to a polypeptide comprising a mutantfragment of Borrelia outer surface protein A (OspA), a nucleic acidencoding the same, a pharmaceutical composition (particularly for use asa medicament or in a method of treating or preventing a Borreliainfection) comprising the polypeptide and/or the nucleic acid, a methodof treating or preventing a Borrelia infection and a method ofimmunizing a subject.

Efforts to develop a subunit vaccine for prevention of Lyme borreliosishave been focused in large part on the use of borrelial outer surfaceprotein A (OspA) as an antigen. The OspA protein is expressed byBorrelia only when it is in the gut of the tick vector. Thus, OspAantibodies produced by vaccination do not fight infection in the body,but rather enter the gut of the tick when it takes a blood meal. There,the antibodies neutralise the spirochetes and block the migration ofbacteria from the midgut to the salivary glands of the tick, the routethrough which Borrelia enters the vertebrate host. Thus, OspA-specificantibodies prevent the transmission of Borrelia from the tick vector tothe human host.

The lipidated form of OspA from B. burgdorferi s.s., strain ZS7,together with aluminium hydroxide was commercially developed as avaccine against Borrelia (LYMErix™) by SmithKline Beecham, nowGlaxoSmithKline (GSK) for the US market. Three doses of LYMErix™ over aperiod of one year were needed for optimal protection. After the firsttwo doses, vaccine efficacy against Lyme borreliosis was 49%, and afterthe third dose 76%. However, shortly after LYMErix™ was commerciallyavailable, it was withdrawn from the market in 2002. Reasons cited werematters of practical application of the vaccine, for example the needfor booster injections every year or every other year, as well as therelatively high cost of this preventive approach compared withantibiotic treatment of early infection. In addition, there was aconcern that LYMErix™ could trigger autoimmune reactions in a subgroupof the population due to sequence homology with a human protein, thoughthis was never proven. In addition, cross-protection against otherclinically important Borrelia species was not provided by this vaccine.

Accordingly, in one embodiment, it was an object of the presentinvention to provide an improved vaccine for the prevention of Lymeborreliosis. Preferably, the vaccine is easily produced while beingprotective, safe and more effective than existing therapies and/orprovides protection against more than one Borrelia species.

The problem underlying the present invention is solved by a polypeptidecomprising a mutant fragment of an outer surface protein A (OspA),wherein the mutant fragment consists of a C-terminal domain of an OspAprotein of Borrelia and differs from the corresponding wild-typefragment by the introduction of at least one disulfide bond.

Surprisingly, it was found that the introduction of at least onedisulfide bond in a mutant fragment increases the protective capacity ofthe polypeptide comprising the mutant OspA fragment relative to apolypeptide comprising the wild-type OspA fragment, as shown in an invivo model of infection. As shown in the Examples, the introduction ofat least one disulfide bond into the B. afzelii OspA C-terminal fragmentincreased its protective capacity relative to the wild-type OspAfragment without a disulfide bond. Tables 2 and 3 provide datademonstrating the protective capacity of mutant fragments with anintroduced disulfide bond (“S2D1-5”) as compared to the wild-type OspAfragment (“S2D0”), as fewer animals were infected after immunizationwith mutant OspA fragments in comparison to wild-type OspA fragments.Some of the mutant OspA fragments tested provided protection comparableto that conveyed by the positive control antigen, the non-lipidatedfull-length OspA protein.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, in a first aspect, the present invention relates to apolypeptide comprising a mutant fragment of an outer surface protein A(OspA), wherein the mutant fragment consists of a C-terminal domain ofan OspA of Borrelia and differs from the corresponding wild-typefragment by the introduction of at least one disulfide bond.

The term B. burgdorferi s.l. encompasses at least 13 Borrelia species(Table A-1). These species occur in different geographic regions, andlive in nature in enzootic cycles involving ticks of the Ixodes ricinuscomplex (also called Ixodes persulcatus complex) and a wide range ofanimal hosts. Four Borrelia species are responsible for the majority ofinfections in humans: B. burgdorferi s.s., B. afzelii, B. bavariensisand B. garinii. Three other species, B. lusitaniae, B. bissettii and B.spielmanii, have occasionally been detected in humans, but their role inLyme borreliosis is uncertain at present. New species of Borrelia arestill being reported.

TABLE A-1 Principal tick vector Location Pathogenic species (4) Borreliaburgdorferi Ixodes scapularis Northeastern/north- (Borrelia burgdorferis.s.) central US Ixodes pacificus Western US Ixodes ricinus EuropeIxodes persulcatus Asia Borrelia garinii Ixodes ricinus Europe Ixodespersulcatus Asia Borrelia afzelii Ixodes ricinus Europe Ixodespersulcatus Asia Borrelia bavariensis Ixodes ricinus Europe Ixodespersulcatus Asia Minimally pathogenic or non- pathogenic species (9)Borrelia andersonii Ixodes dentatus Eastern US Borrelia bissettii Ixodesspinipalpis Western US Ixodes pacificus Europe Ixodes ricinus Borreliavalaisiana Ixodes ricinus Europe and Asia Ixodes columnae Borrelialusitaniae Ixodes ricinus Europe Borrelia spielmanii Ixodes ricinusEurope Borrelia japonica Ixodes ovatus Japan Borrelia tanukii Ixodestanuki Japan Borrelia turdi Ixodes turdus Japan Borrelia sinica Ixodespersulcatus China

As detailed above, Borrelia outer surface protein A (OspA) is anabundant immunogenic lipoprotein of Borrelia of particular interestbecause of its potential as a vaccine candidate. OspA of B. burgdorferis.l. is a basic lipoprotein that has a molecular mass of approximately30 kDa and is encoded on a linear plasmid. An important aspect of theOspA protein is its N-terminal lipidation; that is, the N-terminalcysteine residue is substituted with fatty acids with a chain length ofbetween C14 and C19 with or without double-bonds, a feature thatenhances the immunogenicity of the OspA protein. It has been shown thatpoorly-immunogenic synthetic peptides induce stronger antibody responseswhen lipidated; for example, when covalently coupled to Pam₃Cys (Besslerand Jung, Research Immunology (1992) 143:548-552), a fatty acidsubstitution found at the amino terminus of many bacterial lipoproteinsthat are synthesized with a signal sequence specifying lipid attachment.Additionally, the Pam₃Cys moiety was shown to enhance immune responsesto OspA in mice, partially through its interaction with TLR-2 (Yoder, etal. (2003) Infection and Immunity 71:3894-3900). Therefore, lipidationof a C-terminal fragment of OspA would be expected to enhance theimmunogenicity and protective capacity of the fragment.

Analysis of isolates of B. burgdorferi s.l. obtained in North Americaand Europe has revealed that OspA has antigenic variability and thatseveral distinct groups can be defined based on serology. Anti-OspA mAbswhich bind to specific N- and C-terminal antigenic determinants havebeen reported. Previous studies have shown that the production ofantibodies against the C-terminal epitope LA-2 correlates withprotective immunity after vaccination with OspA (Van Hoecke et al.Vaccine (1996) 14(17-18):1620-6 and Steere et al., N Engl J Med (1998)339:209-215). Antibodies to LA-2 were shown to block the transmission ofBorrelia from tick to host (Golde et al., Infect Immun (1997)65(3):882-889). These studies suggested that the C-terminal portion ofthe OspA protein may be sufficient for inducing protective immunity.Based on information from these and other studies, truncated forms ofOspA comprising the C-terminal portion (also referred to herein as “OspAfragment” or “monomer”) were used in the current invention. Thesetruncated forms of OspA proved to be less protective than thefull-length OspA protein. Surprisingly, however, it was found in thecourse of the current invention that the introduction of a disulfidebond in the truncated form (also referred to herein as “mutant OspAfragment” or “mutant fragment”) overcomes this disadvantage. While notbeing limited to a specific mechanism, it is thought that improvedprotection is due to increased stability of the OspA fragment, as shownin assays measuring thermal stability.

In accordance with the present invention, the mutant OspA fragment maybe derived from any Borrelia species; however, due to their relevance inthe medical field, particularly for humans, B. burgdorferi s.s., B.afzelii, B. bavariensis and B. garinii are preferred. In this regard,these four Borrelia species can be further classified according to theirOspA serotypes, which have been determined by analysis with monoclonalantibodies specific to the respective OspA protein. Serotypes 1-7, whichaccount for the majority of human Borrelia infections, along with theirrates of prevalence, are shown in Table A-2 below.

TABLE A-2 Serotype designation and prevalence of B. burdorferi s.s., B.afzelii, B. bavariensis and B. garinii. Borrelia isolated from humancerebrospinal fluid or skin or from tick vectors were serotyped byprobing whole-cell lysates with mouse monoclonal antibodies, eachspecific to a particular epitope of OspA (as described by Wilske et al.,J. of Clin Microbiol (1993) 31(2): 340-350 and presented by BaxterBioscience at “Climate change effect on ticks and tick-borne diseases”,Brussels, 6 Feb. 2009). OspA serotype Prevalence Strain Seq defined byin human source for ID Borrelia sp. mAb testing disease sequence No: B.burgdorferi s.s. 1 11% B31 20 B. afzelii 2 63% K78 19 B. garinii 3 1.5% PBr 21 B. bavariensis 4  4% PBi 22 B. garinii 5  6% PHEi 23 B. garinii 613% DK29 24 B. garinii 7 0.5%  T25 25

The structure of the OspA protein from B. burgdorferi s.s. strain B31was determined by Li et al. (Proc Natl Acad Sci (1997) 94:3584-3589). Itis composed of N-terminal (β-strands 1 to 4) and central β-sheets(β-strands 5 to 14n [N-terminal part]), barrel sheet 1 (β-strands 14c[C-terminal part] to 16), barrel sheet 2 (β-strands 17 to 21) and aC-terminal α-helix. The term “OspA C-terminal domain” or “C-terminaldomain” or “wild-type fragment” with respect to OspA as used throughoutthe present specification shall mean the C-terminal portion of OspA,i.e., OspA lacking at least the N-terminal β-sheet (including β-strands1 to 4). In OspA from B. burgdorferi s.s. strain B31, the N-terminalsheet consists of amino acids 17 to 70 (following post-translationalcleavage of the 16 aa long lipidation signal peptide). The C-terminalOspA fragment of the current invention may also include a lipidationsignal sequence at the N-terminus, e.g., the lipidation signal sequenceof amino acids 1 to 16 of OspA (SEQ ID NO: 14) or OspB (SEQ ID NO: 15)from B. burgdorferi s.s. strain B31, a lipidation signal sequence fromE. coli, referred to herein as the “lpp lipidation signal” (SEQ ID NO:16), or any other signal sequence, e.g., as defined below.

Lipidation of a protein with an N-terminal lipidation signal sequence,such as those present on a nascent OspA polypeptide, occurs in the E.coli expression vector by the step-wise action of the enzymesdiacylglyceryl transferase, signal peptidase II and transacylase,respectively. The first step is the transfer of a diacylglyceride to thecysteine sulphydryl group of the unmodified prolipoprotein, followed bythe cleavage of the signal peptide by signal peptidase II and, finally,the acylation of the α-amino group of the N-terminal cysteine of theapolipoprotein. The result is the placement of one lipid and a glycerolgroup substituted with two further lipids on the N-terminal cysteineresidue of the polypeptide. The lipidation signal sequence, which iscleaved off during lipidation, is not present in the final polypeptidesequence.

According to the current invention, the mutant OspA fragment may be alipidated protein, also lipoprotein, wherein the lipid moieties, alongwith the glycerol group, is also referred to as “Lip”. According to theinvention, Lip comprises one to three lipids such as C₁₄₋₂₀ alkyl and/orC₁₄₋₂₀ alkenyl attached to a glycerol and the N-terminal cysteine of thepolypeptide of the invention, or preferably wherein Lip is a moiety offormula (I) below,

in which one of R₁, R₂ or R₃ is C₁₄-C₂₀ alkyl or alkenyl, and each ofthe others, independently is C₁₄-C₂₀ alkyl or C₁₄-C₂₀ alkenyl, and X isan amino acid sequence attached to the cysteine residue shown in Formula(I). More preferably, Lip plus the N-terminal cysteine of thepolypeptide is N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy) propylcysteine (referred to herein as “Pam₃Cys” (SEQ ID NO: 139)) and isconnected via the carbonyl C of the cysteine to said amino acid sequenceof the invention. In Formula (I) above R₁, R₂ and R₃ would be palmitoylmoieties and X is an amino acid sequence attached to the cysteineresidue.

In accordance with the current invention, the C-terminal domain of anOspA from a strain other than B. burgdorferi s.s. B31 is defined by (i)lacking at least amino acids 17 to 70 and/or (ii) by lacking at leastthe N-terminal domain homologous to amino acids 17 to 70 of OspA from B.burgdorferi s.s. B31. Additionally, the OspA C-terminal domain accordingto the present invention may also lack further portions of the centralsheet as defined by Li and co-workers (Li et al., supra), particularlyfurther strands such as the amino acid portions from amino acid 17 to82, 93, 105, 118 or 119, preferably 17 to 129, more preferably 1 to 125,1 to 129 or 1 to 130 of any Borrelia, particularly B. burgdorferi s.s.B31, or homologous portions of an OspA protein from a Borrelia sp. otherthan B. burgdorferi s.s. B31.

In the context of the present invention, the OspA C-terminal domain isalso referred to as “OspA fragment” or “fragment of OspA”.

The “mutant fragment” in the context of the polypeptide of the presentinvention and as used throughout the present specification shall meanthe OspA C-terminal fragment, as defined above, which differs from thewild-type fragment by at least two introduced cysteines that can form adisulfide bond. Without being bound to that theory, it is assumed thatthe disulfide bond stabilizes the fragment in a conformation conduciveto the induction of antibody binding. The fold of the wild-typeC-terminal fragment of OspA shows reduced temperature stability incomparison to the full-length protein (Koide et al., Structure-basedDesign of a Second-generation Lyme Disease Vaccine Based on a C-terminalFragment of Borrelia burgdorferi OspA, J. Mol. Biol. (2005)350:290-299). For the present invention, the sequence of the C-terminaldomain of the B. burgdorferi s.s. B31 OspA has been in silico analyzedto determine positions for introduced disulfide bridges that may enhancethe stability of the fold of this C-terminal domain. The results of theanalysis have been transferred to homologous OspA fragments of otherBorrelia species with the assumption that the fold is conserved acrossspecies.

Typically, the disulfide bond may be introduced by introduction of oneor more cysteine residues, wherein a disulfide bond (S—S bridge) isformed between the thiol groups of two cysteine residues. Only onecysteine residue need be introduced if a disulfide bond is formed with acysteine residue present in the wild-type fragment. The one, orpreferably two, cysteine(s) may be introduced by amino acid addition or,preferably, substitution.

The OspA mutant fragment may also comprise further mutations relative tothe wild-type. As detailed above, the structure and surface domain ofOspA are known in the art. Accordingly, the mutant fragment may comprisefurther mutations, particularly at sites not on the surface of theprotein and/or not involved in the immune response and, therefore notimpacting antigenic capacity. These can include one or more amino aciddeletion(s), particularly small (e.g., up to 10 amino acids) deletions,one or more amino acid addition(s) (particularly C- or N-terminally),one or more amino acid substitution(s), particularly one or moreconservative amino acid substitutions. Examples of conservative aminoacid substitutions include, but are not limited to, those listed below:

Ala Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp His Asn;Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Asn Met Leu; Ile Phe Met;Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Preferred mutations include changes in selected portions of thefragment, for example, wherein the sequence with sequence similarity tohuman leukocyte function-associated antigen (hLFA-1), which exists in B.burgdorferi s.s., is modified, for example, replaced by a homologoussequence from an OspA protein from another Borrelia sp. The rationalefor this modification is to reduce the risk for inducing immunologicalcross-reaction with human proteins. Also possible is the addition of asignal sequence for lipidation in the final, or an intermediate,fragment, or the addition of a marker protein (e.g., for identificationor purification).

In some embodiments, the mutant OspA fragment has an amino acid sequencethat has 60%, preferably at least 70%, more preferably at least 80%,more preferably 85%, more preferably 90%, even more preferably 95%sequence identity to the wild-type fragment.

Identity, as known in the art and as used herein, is the relationshipbetween two or more polypeptide sequences, as determined by comparingthe sequences. In the art, identity also means the degree of sequencerelatedness between polypeptide or polynucleotide sequences, as the casemay be, as determined by the match between strings of such sequences.Identity can be readily calculated. While a number of methods exist tomeasure identity between two polynucleotides or two polypeptidesequences, the term is well known to skilled artisans (e.g. SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987).Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity arecodified in computer programs. Preferred computer program methods todetermine identity between two sequences include, but are not limitedto, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN,and FASTA (Altschul, S. et al., 1990).

In contrast to the mutant OspA fragment, the “wild-type fragment” in thecontext of the present invention relates to a fragment of anaturally-occurring OspA of Borrelia. The wild-type fragment is obtainedby N-terminal deletions, but it does not comprise internal deletions(except from signal sequences as detailed herein) or mutations. Inrelation to the mutant OspA fragment, the wild-type fragment consists ofan identical part of the OspA (identical length and same strain of OspA,etc.) and differs only in the mutation(s) detailed above, particularlyin the introduction of at least one disulfide bond or the replacement ofa sequence with human homology.

In one embodiment of the present invention, the mutant OspA fragment maydiffer from the respective wild-type fragment only by the introductionof at least one, preferably exactly one, disulfide bond.

A polypeptide is a single linear polymer of amino acids linked bypeptide bonds. In accordance with the present invention, the polypeptidemay also compromise one or more posttranslational modifications; i.e.,an attached biochemical functional group, such as an attached acetate,phosphate, lipid or carbohydrate, preferably a lipid or lipids attachedto the N-terminal cysteine along with a glycerol, more preferably 1 to 3C₁₄-C₂₀ alkyl or alkenyl moieties, even more preferably 1 to 3 palmitoylgroups, most preferably three palmitoyl groups (Pam₃).

In accordance with the present invention, the polypeptide of the presentinvention comprises the above-described mutant OspA fragment. Accordingto the present invention, it does not comprise (i) the N-terminal sheetas defined above and (ii) optionally further strands of the centralsheet as defined above. However, the polypeptide may comprise one ormore functional sequences such as a signal sequence, e.g., a lipidationsignal sequence or a posttranslational modification, such as lipidation.

In a further embodiment of the present invention, the polypeptide of thepresent invention consists of (i) one or more mutant OspA fragments,optionally joined by linkers, e.g., as defined below and (ii) optionallyone or more amino acids heterologous to OspA, particularly a signalsequence and (iii) optionally a posttranslational modification, such aslipidation.

The polypeptide of the present invention has protective capacity. Asdetailed above, the introduction of a disulfide bond into the mutantOspA fragment increases the protective capacity of the polypeptiderelative to a polypeptide comprising the respective fragment without thedisulfide bond(s). In some embodiments, the protective capacity isincreased by at least 10%, more preferably by at least 20%, morepreferably by at least 30%, more preferably by at least 40%, morepreferably by at least 50%, more preferably by at least 60%, morepreferably by at least 70%, more preferably by at least 80%, even morepreferably by at least 90%.

The term protective capacity describes the ability to protect a subjectagainst a Borrelia infection. With respect to the polypeptide of theinvention, protective capacity relates to the ability of the polypeptideto induce an immune response that protects a subject against a Borreliainfection. Protective capacity can be tested by administering to asubject the polypeptide in a manner to induce an immune reaction againstthe polypeptide. Thereafter, the subject may be challenged withBorrelia. The subject's reaction to the infection is monitored.Particularly, the presence of Borrelia in the subject may be determined.For example, the polypeptide is protective if Borrelia cannot bedetected in the subject. The presence of Borrelia can be determined bydetecting Borrelia-specific nucleic acids (e.g., by PCR) orBorrelia-specific antibodies (e.g., by ELISA or Western blot) or bydetecting Borrelia itself (e.g., culturing organs or tissues in growthmedium and verifying the presence of Borrelia by microscopy). Inparticular, the protective capacity (“pc”), reported as a percentage,for a particular dose is defined as follows:

pc (%)=[(number of total tested subjects−number of Borrelia-infectedsubjects)/number of total tested subjects]×100

Differences in protective capacity (Δpc) may be determined by, e.g.comparing the protective capacity (pc) of a mutant OspA fragment with adisulfide bond(s) (pc [with bond]) to the protective capacity of an OspAfragment without a disulfide bond(s) (pc [w/o bond]). In accordance withthe present invention, the polypeptides to be compared differ only inthe introduction of at least one disulfide bond. The change inprotective capacity (Δpc) by the introduction of the disulfide bond(s)is determined as follows:

Δpc=(pc[sample]−pc[control])

e.g. Δpc=(pc[with bond]−pc[w/o bond])

If Δpc is greater than zero (>0), assuming all other parameters (e.g.,dose and assay) are the same, then the protective capacity of the sample(e.g. the mutant OspA fragment with a disulfide bond(s)) is better thanthe protective capacity of the control (e.g. the OspA fragment without adisulfide bond(s)). Conversely, if Δpc is less than zero (<0) andassuming all other parameters (e.g., dose and assay) are the same, thenthe protective capacity of the sample (e.g. the mutant OspA fragmentwith a disulfide bond(s)) is less than the protective capacity of thecomparison (e.g., the OspA fragment without a disulfide bond(s)).

Preferably, the polypeptide of the present invention is assessed for itsprotective capacity by an in vivo animal assay wherein mice immunizedwith the polypeptide of the invention or with a control inoculate arechallenged with Borrelia introduced into the immunized subjects with ahypodermic needle. More preferably, the polypeptide of the presentinvention is assessed for its protective capacity by an in vivo animalassay comprising the steps of a) applying at least one I. ricinus nymphinfected with Borrelia, e.g., B. afzelii, strain IS1, to a mouse that isimmunized with said first polypeptide of the first aspect; and b)applying at least one I. ricinus nymph infected with Borrelia, e.g., B.afzelii, strain IS1, to a second mouse that is immunized with saidsecond polypeptide of the first aspect; and c) comparing the rates ofinfection in the two mice. Preferably, the assay or test is done with agroup of mice per polypeptide to be tested. A suitable test is alsodescribed and illustrated in the Examples.

In a preferred embodiment of the present invention, the products of theinvention such as, e.g. the polypeptides of the invention comprising themutant OspA fragment with a disulfide bond(s) administered 3 times at adose of 5.0 μg, preferably 1.0 μg, preferably 0.3 μg or lower have aprotective capacity of 80% or more, preferably 90% or more, even morepreferably 95% or more, most preferred 99% or more. It has beensurprisingly observed that immunization with an OspA mutant fragment ofone Borrelia serotype can provide cross-protection against other anotherserotype (Example 4, Table 4). Based on this finding, it might beanticipated that the dose of polypeptide of the present invention couldbe even further reduced.

In a preferred embodiment of the present invention, the C-terminaldomain of an OspA protein of Borrelia consists of (i) the amino acidsfrom position 126, 131 or 130 to position 273 of the OspA of B. afzelii,strain K78 or (ii) the homologous domain to amino acids of OspA from aBorrelia strain other than B. afzelii, strain K78. Accordingly, thepolypeptide of the present invention comprises or consists of (i) one ormore of these mutant fragments, optionally joined by linkers, e.g., asdefined below and (ii) optionally one or more amino acids heterologousto OspA, particularly a signal sequence or site for a post-translationalmodification such as lipidation and (iii) optionally a posttranslationalmodification, such as lipidation.

In accordance with the present invention, a disulfide bond is introducedinto an OspA fragment. This may preferably be achieved by introducinginto the fragment at least 1 or 2 cysteine(s), particularly 2 cysteines,in order to allow for the formation of the at least one disulfide bond.Only one cysteine may be introduced, if another cysteine in the fragmentis available for a disulfide bond. However, preferably two cysteines areintroduced. The cysteine(s) is/are introduced by amino acid addition orsubstitution, preferably substitution. In case of addition, the cysteineis inserted into the amino acid sequence between two amino acids,whereas in case of substitution one amino acid is replaced with thecysteine.

In accordance with the present invention, the OspA may be from anyBorrelia strain, particularly from those specified herein such as B.burgdorferi s.s., B. garinii, B. afzelii, B. andersonii, B. bissettii,B. valaisiana, B. lusitaniae, B. spielmanii, B. japonica, B. tanukii, B.turdi or B. sinica, B. bavariensis, preferably from B. burgdorferi s.s.,B. afzelii, B. bavariensis or B. garinii. Preferably, the OspA is fromB. afzelii, particularly strain K78, OspA serotype 2 (SEQ ID NO: 19); B.burgdorferi s.s., particularly strain B31, OspA serotype 1 (SEQ ID NO:20); B. garinii, particularly strain PBr, OspA serotype 3 (SEQ ID NO:21); B. bavariensis, particularly strain PBi, OspA serotype 4 (SEQ IDNO: 22); B. garinii, particularly strain PHei, OspA serotype 5 (SEQ IDNO: 23); B. garinii, particularly strain DK29, OspA serotype 6 (SEQ IDNO: 24) or B. garinii, particularly strain T25, OspA serotype 7 (SEQ IDNO: 25). The amino acid sequences of these OspA proteins (full-length)are given below.

TABLE A-3 Accession numbers of OspA sequences from selected strains ofBorrelia species. Organism_Strain db|accession.version Organism_Straindb|accession.version Organism_Strain db|accession.version Bbu_156a(serotype 1) gb|ACL33776.1 Bbu_K48 emb|CAA44492.1 Bga_Mng4702gb|ABF29559.1 Baf_K78 (serotype 2) emb|CAA49828.1 Bbu_N40 gb|ACS94765.1Bga_N34 emb|CAB64763.1 Bga_PBr (serotype 3) emb|CAA56549.1 Bbu_P0A3N6.1sp|P0A3N6.1 Bga_Nov1006 gb|ACD02016.1 Bga_PBi (serotype 4)emb|CAA56550.1 Bbu_PBo emb|CAA56468.1 Bga_Nov105 gb|ABF29551.1 Bbu_PHei(serotype 5) tr|Q06228 Bbu_PBre emb|CAA59742.1 Bga_Nov14506gb|ACD02013.1 Bbu_DK29 (serotype 6) emb|CAA45010.1 Bbu_PHeiemb|CAA56544.1 Bga_Nov14606 gb|ACD02017.1 Bga_T25 (serotype 7)emb|CAA56547.1 Bbu_PKa emb|CAA56467.1 Bga_Nov2005 gb|ABF29553.1Baf_ACA-1 gb|ACJ73559.1 Bbu_PKo emb|CAA46550.1 Bga_Nov2006 gb|ACD02018.1Baf_K78 (sequenced) Bbu_Poti_B1 emb|CAB64754.1 Bga_Nov3305 gb|ABF29554.1Baf_Khab_625 gb|AAR96311.1 Bbu_Poti_B2 emb|CAB64755.1 Bga_Nov405gb|ABF29552.1 Baf_Khab2-Sakh gb|AAP94134.1 Bbu_Poti_B3 emb|CAB64756.1Bga_Nov7006 gb|ACD02014.1 Baf_Khab470 gb|AAO91923.1 Bbu_PTroemb|CAA56471.1 Bga_Nov9906 gb|ACD02015.1 Baf_Khab505 gb|AAO91925.1Bbu_PWudI emb|CAA56469.1 Bga_PBi gb|AAT93773.1 Baf_LU192 (sequenced,partial) Bbu_PWudI/6 emb|CAA56470.1 Bga_PBr emb|CAA56549.1 Baf_Mng3602gb|ABF29573.1 Bbu_PWudII emb|CAA56546.1 Bga_Q1HLH6 gb|ABF29564.1Baf_Mng4302 gb|ABF29574.1 Bbu_Q04851.1 sp|Q04851.1 Bga_T25emb|CAA56547.1 Baf_Mng6702 gb|ABF29578.1 Bbu_Q04968.1 sp|Q04968.1Bga_TIsl emb|CAA59727.1 Baf_Mng702 gb|ABF29572.1 Bbu_Q09086.1sp|Q09086.1 Bga_TN emb|CAA56545.1 Baf_Nov1105 gb|ABF29569.1 Bbu_Q09087.1sp|Q09087.1 Bga_Tom1003 gb|ABF29564.1 Baf_Nov11506 gb|ACD02019.1Bbu_Q44738 tr|Q44738 Bga_Tom1805 gb|ABF29567.1 Baf_Nov3005 gb|ABF29570.1Bbu_Q44956 emb|CAA56937.1 Bga_Tom203 gb|ABF29562.1 Baf_P0A3N7.1sp|P0A3N7.1 Bbu_Q44962 dbj|BAA06133.1 Bga_Tom2903 gb|ABF29565.1 Baf_PHoemb|CAA59724.1 Bbu_Q45039 emb|CAR95556.1 Bga_Tom3005 gb|ABF29568.1Baf_PKo gb|ABH02138.1 Bbu_Q45040 tr|Q45040 Bga_Tom303 gb|ABF29563.1Baf_PLe emb|CAA59970.1 Bbu_S-1-10 gb|AAB96354.1 Bga_Tom3101gb|ABF29557.1 Baf_PLj7 emb|CAA59725.1 Bbu_T.R.O. emb|CAA46549.1Bga_Tom3803 gb|ABF29566.1 Baf_PLud emb|CAA59726.1 Bbu_T255emb|CAA59730.1 Bga_Tom5102 gb|ABF29560.1 Baf_Tom1103 gb|ABF29581.1Bbu_UK emb|CAB64758.1 Bga_Tom5202 gb|ABF29561.1 Baf_Tom1303gb|ABF29582.1 Bbu_VS116 emb|CAB64757.1 Bga_Tom7105 gb|ABF29556.1Baf_Tom1503 gb|ABF29583.1 Bbu_VS461 emb|CAA82329.1 Bga_VS100emb|CAB64765.1 Baf_Tom2303 gb|ABF29584.1 Bbu_WI91-23 ref|ZP_03091138.1Bga_VS307 emb|CAB64764.1 Baf_Tom2403 gb|ABF29585.1 Bbu_ZQ1emb|CAA01704.1 Bga_WABSou emb|CAA59728.1 Baf_Tom2504 gb|ABF29577.1Bbu_ZS7 gb|ACK74228.1 Bja_Cow611 emb|CAB64759.1 Baf_Tom2803gb|ABF29586.1 Bga_BgVir-1 gb|ABF29555.1 Bja_F63 emb|CAB64760.1Baf_Tom3401 gb|ABF29571.1 Bga_Far04 ref|ZP_03328706.1 Bja_HO14emb|CAB64762.1 Baf_Tom3703 gb|ABF29587.1 Bga_FujiP2 gb|AAA92301.1Bja_IKA2 emb|CAB64761.1 Baf_Tom4703 gb|ABF29588.1 Bga_IP90emb|CAJ75754.1 Blu_A8D057 gb|ABR22627.1 Baf_Tom5403 gb|ABF29575.1Bga_Ip90 emb|CAJ75754.1 Blu_A8D060 gb|ABR22625.1 Baf_Tom603gb|ABF29579.1 Bga_JEM1 gb|AAB81567.1 Blu_A8D075 gb|ABR22628.1Baf_Tom6303 gb|ABF29576.1 Bga_JEM2 gb|AAB81569.1 Blu_A8D079gb|ABR22629.1 Baf_Tom703 gb|ABF29580.1 Bga_JEM3 gb|AAB81571.1Blu_ABR22624.1 gb|ABR22624.1 Baf_XJ23 gb|AAB95225.1 Bga_JEM4dbj|BAA19222.1 Blu_ABR22S26.1 gb|ABR22626.1 Bbu_118a ref|ZP_02720644.1Bga_JEM5 gb|AAB81573.1 Bsp_A14S gb|AAD16455.1 Bbu_156a gb|ACL33776.1Bga_JEM6 gb|AAB81575.1 Btu_Ya501 dbj|BAA32513.1 Bbu_19857 emb|CAA48196.1Bga_JEM7 gb|AAB81577.1 Bva_AR-2 gb|AAF00571.1 Bbu_2005348A prf|2005348ABga_JEM8 gb|AAB81579.1 Bva_M19 gb|AAF00573.1 Bbu_2005348B prf|2005348BBga_Khab3155 gb|AAR96310.1 Bva_M49 gb|AAF00574.1 Bbu_297 emb|CAA59729.1Bga_Khab550 gb|AAR96306.1 Bva_M52 gb|AAF00575.1 Bbu_29805ref|ZP_03092996.1 Bga_Khab616 gb|AAR96307.1 Bva_M53 gb|AAF00576.1Bbu_64b ref|ZP_03097520.1 Bga_Khab648 gb|AAR96308.1 Bva_M7 gb|AAF00572.1Bbu_72a ref|ZP_02724465.1 Bga_Khab722 gb|AAR96309.1 Bva_Q9RM88emb|CAB56150.1 Bbu_80a ref|ZP_03088001.1 Bga_Khab23 gb|AAP94125.1Bva_QLZSP1 gb|ACA13516.1 Bbu_94a ref|ZP_02725946.1 Bga_Khab24gb|AAP94126.1 Bva_QSDS4 gb|ACA13517.1 Bbu_AAB23809.1 gb|AAB23809.1Bga_Khab31 gb|AAP94127.1 Bva_QSYSP3 gb|ACA13518.1 Bbu_AAB23810.1gb|AAB23810.1 Bga_Khab31a gb|AAP94128.1 Bva_QSYSP4 gb|ACA13519.1 Bbu_B29gb|AAA18508.1 Bga_Khab-466 gb|AAP94129.1 Bva_QTMP2 gb|ACA13520.1 Bbu_B31gb|AAC66260.1 Bga_Khab489 gb|AAP94130.1 Bva_QX-S13 gb|ACA13521.1Bbu_Bol26 ref|ZP_02531917.1 Bga_Khab5-Sakh gb|AAO91932.1 Bva_UKgb|AAF00570.1 Bbu_C-1-11 gb|AAB96351.1 Bga_Khab506 gb|AAP94132.1Bva_VS116 gb|AAF00569.1 Bbu_CA-11.2a_1 ref|ZP_03094587.1 Bga_Khab516gb|AAP94133.1 Bsp_10MT dbj|BAA32516.1 Bbu_CA-11.2a_2 ref|ZP_03094587.1Bga_Khab721 gb|AAP94131.1 Bsp_5MT dbj|BAA32515.1 Bbu_CA-11.2a_CA-112aref|ZP_03094587.1 Bga_Khab2119 gb|AAO91928.1 Bsp_Am501 dbj|BAA32514.1Bbu_CAA00316.1 emb|CAA00316.1 Bga_Khab2559 gb|AAO91929.1 Bsp_LV5gb|AAB96353.1 Bbu_CAA42842.1 emb|CAA42842.1 Bga_Khab2560 gb|AAO91930.1Bsp_PAnz emb|CAJ43585.1 Bbu_CAA44258.1 emb|CAA44258.1 Bga_Khab2594gb|AAO91931.1 Bsp_PHaP_PHap emb|CAJ43582.1 Bbu_CAR95597.1 emb|CAR95597.1Bga_Khab430 gb|AAO91919.1 Bsp_PJes emb|CAJ43586.1 Bbu_DK1 gb|AAA22955.1Bga_Khab448 gb|AAO91920.1 Bsp_PMai emb|CAJ43584.1 Bbu_DK29emb|CAA45010.1 Bga_Khab457 gb|AAO91921.1 Bsp_PMew emb|CAJ43583.1Bbu_DK6_Danish_isolate emb|CAA58601.1 Bga_Khab468 gb|AAO91922.1Bsp_PSigII emb|CAJ43581.1 Bbu_G2 gb|AAA88846.1 Bga_Khab492 gb|AAO91924.1Bsp_SV1 ref|ZP_03095680.1 Bbu_G25 emb|CAA82328.1 Bga_Khab511gb|AAO91926.1 Bbi_25015 gb|AAB21761.1 Bbu_H.E. emb|CAA46551.1Bga_Khab560 gb|AAO91927.1 Bbi_DN127 emb|CAB64766.1 Bbu_HB19gb|AAC18776.1 Bga_LV4 gb|AAB96352.1 Bbi_Q09087.1 gb|AAB21761.1Abbreviations: Baf = Borrelia afzelii, Bbu = Borrelia burgdorferi s.s.,Bga = Borrelia garinii, Bsp = Borrelia spielmanii, Bbi = Borreliabissettii, Bva = Borrelia valaisiana, Btu = Borrelia turicatae, Bdu =Borrelia duttonii, Blu = Borrelia lusitaniae, Bja = Borrelia japonica,gb = GenBank, emb = EMBL, tr = UniProt/tremble, sp = UniProt/Swissprot,prf = Protein Research Foundation, dbj = DNA Databank of Japan (DDBJ),pdb = Protein Data Bank, db = database

In accordance with the present invention, the disulfide bond may beformed between cysteines that have been introduced at any position ofthe OspA fragment allowing or supporting appropriate folding of thefragment. The positions may be selected, as detailed above, based on theknown structure of the OspA. In a preferred embodiment, the polypeptideof the current invention contains at least one disulfide bond betweenany of positions 182+/−3 and any of positions 269+/−3 (disulfide bondtype 1); any of positions 182+/−3 and any of positions 272+/−3(disulfide bond type 2); any of positions 244+/−3 and any of positions259+/−3 (disulfide bond type 3); any of positions 141+/−3 and any ofpositions 241+/−3 (disulfide bond type 4); any of positions 165+/−3 andany of positions 265+/−3 (disulfide bond type 5); any of positions185+/−3 and any of positions 272+/−3 (disulfide bond type 6); any ofpositions 199+/−3 and any of positions 223+/−3 (disulfide bond type 7);any of positions 243+/−3 and any of positions 262+/−3 (disulfide bondtype 8); any of positions 184+/−3 and any of positions 204+/−3(disulfide bond type 9); any of positions 201+/−3 and any of positions214+/−3 (disulfide bond type 10); any of positions 246+/−3 and any ofpositions 259+/−3 (disulfide bond type 11); and/or any of positions167+/−3 and any of positions 178+/−3 (disulfide bond type 12) of a B.afzelii, particularly B. afzelii K78 serotype 2 OspA, or the homologousamino acids of an OspA from a Borrelia sp. other than B. afzelii, suchas B. burgdorferi s.s., particularly strain B31, serotype 1; B. garinii,particularly strain PBr, serotype 3; B. bavariensis, particularly strainPBi, serotype 4; B. garinii, particularly strain PHei, serotype 5; B.garinii, particularly strain DK29, serotype 6 or B. garinii,particularly strain T25, serotype 7.

More particularly, the polypeptide of the current invention contains theat least one disulfide bond between any of positions 182 and 269(disulfide bond type 1); positions 182 and 272 (disulfide bond type 2);positions 244 and 259 (disulfide bond type 3); positions 141 and 241(disulfide bond type 4); positions 165 and 265 (disulfide bond type 5);positions 185 and 272 (disulfide bond type 6); positions 199 and 223(disulfide bond type 7); positions 243 and 262 (disulfide bond type 8);positions 184 and 204 (disulfide bond type 9); positions 201 and 214(disulfide bond type 10); positions 246 and 259 (disulfide bond type11); and/or positions 167 and 178 (disulfide bond type 12) of a B.afzelii, particularly B. afzelii K78 serotype 2 OspA, or the homologousamino acids of an OspA from a Borrelia other than B. afzelii, such as B.burgdorferi s.s., particularly strain B31, serotype 1; B. garinii,particularly strain PBr, serotype 3; B. bavariensis, particularly strainPBi, serotype 4; B. garinii, particularly strain PHei, serotype 5; B.garinii, particularly strain DK29, serotype 6 or B. garinii,particularly strain T25, serotype 7.

TABLE A-4 Disulfide bond types with nomenclature and the position of thecysteine substitutions in the serotype 2 OspA protein. Position ofcysteines in B. afzelii Disulfide bond type Nomenclature K78 serotype 2OspA wild-type sequence D0 No cysteine substitutions 1 D1 182 and 269 2D2 182 and 272 3 D3 244 and 259 4 D4 141 and 241 5 D5 165 and 265 6 D6185 and 272 7 D7 199 and 223 8 D8 243 and 262 9 D9 184 and 204 10 D10201 and 214 11 D11 246 and 259 12 D12 167 and 178

Even more preferred are disulfide bond types 1 to 5, especiallydisulfide bond types 1 to 4.

It is noted that:

Position 182+/−3 is an abbreviation for position 179, 180, 181, 182,183, 184 or 185, preferably 182.

Position 269+/−3 is an abbreviation for position 266, 267, 268, 269,270, 271 or 272, preferably 269.

Position 272+/−3 is an abbreviation for position 269, 270, 271, 272,273, 274 or 275, preferably 272.

Position 244+/−3 is an abbreviation for position 241243, 242, 243, 244,245, 246 or 247, preferably 244.

Position 259+/−3 is an abbreviation for position 256, 257, 258, 259,260, 261 or 262, preferably 259.

Position 141+/−3 is an abbreviation for position 138, 139, 140, 141,142, 143 or 144, preferably 141.

Position 241+/−3 is an abbreviation for position 238, 239, 240, 241,242, 243 or 244, preferably 241.

Position 165+/−3 is an abbreviation for position 162, 163, 164, 165,166, 167 or 168, preferably 165.

Position 265+/−3 is an abbreviation for position 262, 263, 264, 265,266, 267 or 268, preferably 265.

Position 185+/−3 is an abbreviation for position 182, 183, 184, 185,186, 187 or 188, preferably 185.

Position 199+/−3 is an abbreviation for position 196, 197, 198, 199,200, 201 or 202, preferably 199.

Position 223+/−3 is an abbreviation for position 220, 221, 222, 223,224, 225 or 226, preferably 223.

Position 243+/−3 is an abbreviation for position 240, 241, 242, 243,244, 245 or 246, preferably 143.

Position 262+/−3 is an abbreviation for position 259, 260, 261, 262,263, 264 or 265, preferably 262.

Position 184+/−3 is an abbreviation for position 181, 182, 183, 184,185, 186 or 187, preferably 184.

Position 204+/−3 is an abbreviation for position 201, 202, 203, 204,205, 206 or 207, preferably 204.

Position 201+/−3 is an abbreviation for position 198, 199, 200, 201,202, 203 or 204, preferably 201.

Position 214+/−3 is an abbreviation for position 211, 212, 213, 214,215, 216 or 217, preferably 214.

Position 246+/−3 is an abbreviation for position 243, 244, 245, 246,247, 248 or 249, preferably 246.

Position 167+/−3 is an abbreviation for position 164, 165, 166, 167,168, 169 or 170, preferably 167.

Position 178+/−2+/−3 is an abbreviation for position 175, 176, 177, 178,179, 180 or 181, preferably 178.

In a preferred embodiment, the mutant fragment is derived from the aminoacids from position 126, 130 or 131 to position 273 of the wild-typesequence of the OspA of B. afzelii strain K78, serotype 2 (SEQ ID NO:19) and differs only by the introduction of at least one disulfide bond,particularly wherein the at least one disulfide bond is betweenpositions 182 and 269 (disulfide bond type 1); positions 182 and 272(disulfide bond type 2); positions 244 and 259 (disulfide bond type 3);positions 141 and 241 (disulfide bond type 4); positions 165 and 265(disulfide bond type 5); positions 185 and 272 (disulfide bond type 6);positions 199 and 223 (disulfide bond type 7); positions 243 and 262(disulfide bond type 8); positions 184 and 204 (disulfide bond type 9);positions 201 and 214 (disulfide bond type 10); positions 246 and 259(disulfide bond type 11); and/or positions 167 and 178 (disulfide bondtype 12), or the homologous fragments and positions of an OspA from aBorrelia sp. other than B. afzelii, such as B. burgdorferi s.s.,particularly strain B31, serotype 1; B. garinii, particularly strainPBr, serotype 3; B. bavariensis, particularly strain PBi, serotype 4; B.garinii, particularly strain PHei, serotype 5; B. garinii, particularlystrain DK29, serotype 6 or B. garinii, particularly strain T25, serotype7.

In a still more preferred embodiment, the mutant fragment has an aminoacid sequence selected from the group consisting of SEQ ID NO: 167, SEQID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO:172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQID NO: 177, SEQ ID NO: 178 and an amino acid sequence that has 80%, morepreferably 85%, more preferably 90%, even more preferably 95% sequenceidentity to at least one of sequences with SEQ ID NOs: 2 to 13, whereinthe cysteines are not replaced. Further details on mutations andsequence identity are given above.

As detailed above, the polypeptide of the present invention may comprisesignal sequences. It has been shown that lipidation confers adjuvantproperties on OspA. Accordingly, lipidated forms of the polypeptide ofthe invention or polypeptides comprising a lipidation signal arepreferred. In a preferred embodiment, the polypeptide of the currentinvention comprises a lipidation signal, preferably a lipidation signalof a Borrelia outer surface protein, OspA or OspB (SEQ ID NOs: 14 and15, respectively) or more preferably an E. coli lpp lipidation signalsequence (SEQ ID NO: 16). The OspA fragment of the invention comprisinga lipidation signal is lipidated during processing and the lipidationsignal peptide is cleaved off. Therefore the signal peptide is no longerpresent in the mature lipidated protein.

Lipidated proteins according to the current invention are labeled with“Lip” at the N-terminus to indicate the addition of 3 fatty acid groupsand a glycerol to the polypeptide (see FIG. 3). Suitable lipidationsignals as described above include MKKYLLGIGLILALIA (SEQ ID NO: 14),MRLLIGFALALALIG (SEQ ID NO: 15) and MKATKLVLGAVILGSTLLAG (SEQ ID NO:16). Because lipid moieties and a glycerol are attached to theN-terminal cysteine residue which is present in the full-lengthwild-type OspA protein, OspA C-terminal fragments for lipidation mayadditionally comprise a peptide comprising a cysteine residue followedby additional amino acids, herein referred to as “Lipidation Peptide” or“LP” (see FIGS. 1 and 2). For example, sequences such as CSS (SEQ ID NO:210) or CKQN (SEQ ID NO: 211) immediately C-terminal to the lipidationsignal sequence provide an N-terminal cysteine residue for lipidationupon cleavage of the lipidation signal peptide. The lipidatedcysteine-containing peptides are present in the final lipidatedpolypeptide of the invention.

It has been found that the OspA protein of B. burgdorferi s.s. comprisesa sequence with the capacity to bind to a T-cell receptor that also hasthe capacity to bind to human leukocyte function-associated antigen(hLFA-1) (herein referred to also as “hLFA-1-like sequence”). Thesimilarity of this OspA region to hLFA-1 may result in an immuneresponse with cross-reactivity upon administration of B. burgdorferis.s. OspA to a human subject and may induce autoimmune diseases,particularly autoimmune arthritis, in susceptible individuals.Accordingly, in a preferred embodiment, the polypeptide of the currentinvention does not comprise a sequence with binding capacity to theT-cell receptor that has a binding capacity to the human leukocytefunction-associated antigen (hLFA-1), and particularly does not comprisethe amino acid sequence GYVLEGTLTAE (SEQ ID NO: 17). To this end, thehLFA-1-like sequence, particularly the amino acid sequence GYVLEGTLTAE(SEQ ID NO: 17), may be replaced with a homologous sequence from an OspAprotein of another Borrelia sp., particularly with NFTLEGKVAND (SEQ IDNO: 18).

In a preferred embodiment, the polypeptide of the current inventioncomprising at least one disulfide bond essentially establishes the sameprotective capacity with said polypeptide against a Borrelia infectionrelative to at least one of the wild-type full-length OspA proteinsderived from at least one Borrelia strain, particularly B. afzelii K78,OspA serotype 2 (SEQ ID NO: 19); B. burgdorferi s.s., particularlystrain B31, serotype 1 (SEQ ID NO: 20); B. garinii, particularly strainPBr, serotype 3 (SEQ ID NO: 21); B. bavariensis, particularly strainPBi, serotype 4 (SEQ ID NO: 22); B. garinii, particularly strain PHei,serotype 5 (SEQ ID NO: 23); B. garinii, particularly strain DK29,serotype 6 (SEQ ID NO: 24) or B. garinii, particularly strain T25,serotype 7 (SEQ ID NO: 25).

In order to provide cross-protection against different Borrelia speciesor OspA serotypes, the development of a multivalent vaccine isdesirable. Accordingly, in another preferred embodiment, the polypeptideof the first aspect comprises at least two mutant fragments from twodifferent Borrelia serotypes as defined above. In a preferredembodiment, the polypeptide of the first aspect comprises at least twomutant OspA fragments which are selected from the group consisting of

-   -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 2;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 3;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 4;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 5;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 6;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 7;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 8;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 1 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 3;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 4;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 5;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 6;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 7;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 8;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 2 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 4;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 5;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 6;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 7;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 8;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 3 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 5;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 6;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 7;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 8;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 4 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 5 and fragment with disulfide        bond type 6;    -   fragment with disulfide bond type 5 and fragment with disulfide        bond type 7;    -   fragment with disulfide bond type 5 and fragment with disulfide        bond type 8;    -   fragment with disulfide bond type 5 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 5 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 5 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 5 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 6 and fragment with disulfide        bond type 7;    -   fragment with disulfide bond type 6 and fragment with disulfide        bond type 8;    -   fragment with disulfide bond type 6 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 6 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 6 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 6 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 7 and fragment with disulfide        bond type 8;    -   fragment with disulfide bond type 7 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 7 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 7 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 7 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 8 and fragment with disulfide        bond type 9;    -   fragment with disulfide bond type 8 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 8 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 8 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 9 and fragment with disulfide        bond type 10;    -   fragment with disulfide bond type 9 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 9 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 10 and fragment with disulfide        bond type 11;    -   fragment with disulfide bond type 10 and fragment with disulfide        bond type 12;    -   fragment with disulfide bond type 11 and fragment with disulfide        bond type 12;    -   and

particularly wherein

-   -   the fragment with disulfide bond type 1 has the amino acid        sequence of SEQ ID NO: 2 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 2, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 2 has the amino acid        sequence of SEQ ID NO: 3 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 3, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 3 has the amino acid        sequence of SEQ ID NO: 4 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 4, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 4 has the amino acid        sequence of SEQ ID NO: 5 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 5, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 5 has the amino acid        sequence of SEQ ID NO: 6 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 6, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 6 has the amino acid        sequence of SEQ ID NO: 7 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 7, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 7 has the amino acid        sequence of SEQ ID NO: 8 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 8, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 8 has the amino acid        sequence of SEQ ID NO: 9 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 9, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 9 has the amino acid        sequence of SEQ ID NO: 10 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 10, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 10 has the amino acid        sequence of SEQ ID NO: 11 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 11, wherein the        cysteines are not replaced;    -   the fragment with disulfide bond type 11 has the amino acid        sequence of SEQ ID NO: 12 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 12, wherein the        cysteines are not replaced; and/or    -   the fragment with disulfide bond type 12 has the amino acid        sequence of SEQ ID NO: 13 or an amino acid sequence that has at        least 80%, more preferably 85%, more preferably 90%, even more        preferably 95% sequence identity to SEQ ID NO: 13, wherein the        cysteines are not replaced.

Please note that further details on mutations and sequence identity aregiven above.

TABLE A-5 Nomenclature and SEQ ID NOs. of mutant OspA fragmentheterodimers, non-lipidated and lipidated, described in the currentinvention. SEQ ID NO: Mutant OspA fragment heterodimer* S1D4-S2D4 43S1D1-S2D1 47 S3D4-S4D4 51 S3D1-S4D1 55 S5D4-S6D4 59 S5D1-S6D1 63S2D4-S1D4 67 S2D1-S1D1 71 S4D4-S3D4 75 S4D1-S3D1 79 S6D4-S5D4 83S6D1-S5D1 87 S1D4-S2D1 91 S1D1-S2D4 95 S3D4-S4D1 99 S3D1-S4D4 103S5D4-S6D1 107 S5D1-S6D4 111 S2D4-S1D1 115 S2D1-S1D4 119 S4D4-S3D1 123S4D1-S3D4 127 S6D4-S5D1 131 S6D1-S5D4 135 Lipidated mutant OspA fragmentheterodimer* Lip-S1D4-S2D4 185 Lip-S1D1-S2D1 186 Lip-S3D4-S4D4 187Lip-S3D1-S4D1 188 Lip-S5D4-S6D4 189 Lip-S5D1-S6D1 190 Lip-S2D4-S1D4 191Lip-S2D1-S1D1 192 Lip-S4D4-S3D4 193 Lip-S4D1-S3D1 194 Lip-S6D4-S5D4 195Lip-S6D1-S5D1 196 Lip-S1D4-S2D1 197 Lip-S1D1-S2D4 198 Lip-S3D4-S4D1 199Lip-S3D1-S4D4 200 Lip-S5D4-S6D1 201 Lip-S5D1-S6D4 202 Lip-S2D4-S1D1 203Lip-S2D1-S1D4 204 Lip-S4D4-S3D1 205 Lip-S4D1-S3D4 206 Lip-S6D4-S5D1 207Lip-S6D1-S5D4 208 *S = Serotype (1-6) (see Table A-2); D = DisulfideBond Type (see Table A-4); Lip = lipidation: the N-terminal addition ofglycerol and fatty acid residues.

In another preferred embodiment, the polypeptide according to the firstaspect comprises at least two or three mutant fragments which areconnected via one or more linkers. A linker is a rather short amino acidsequence employed to connect two fragments. It should be designed inorder to avoid any negative impact on the fragments, their interactionin subjects to be treated or vaccinated or upon their protectivecapacity. Preferred are short linkers of at most 21 amino acids,particularly at most 15 amino acids, especially at most 12 or 8 aminoacids. More preferably, the one or more linkers is/are composed of smallamino acids in order to reduce or minimize interactions with thefragments, such as glycine, serine and alanine. Examples or preferredlinkers include linkers comprising or consisting of polyG, such as (G)₃(SEQ ID NO: 36), (G)₁₂ (SEQ ID NO: 37), GAGA (SEQ ID NO: 38), (GAGA)₂(SEQ ID NO: 39), (GAGA)₃ (SEQ ID NO: 40), (GGGS)₂ (SEQ ID NO: 41), or(GGGS)₃ (SEQ ID NO: 42). A more preferred linker is the “LN1 peptidelinker”, a fusion of two separate loop regions of the N-terminal half ofOspA from B. burgdorferi s.s., strain B31 (aa 65-74 and aa 42-53, withan amino acid exchange at position 53 of D53S) which has the followingsequence: GTSDKNNGSGSKEKNKDGKYS (SEQ ID NO: 184).

In another preferred embodiment, the polypeptide according to the firstaspect comprises a polypeptide with a total size of at most 500 aminoacids, comprising two or three different mutant fragments as defined inpreferred embodiments of the first aspect; or a polypeptide whichconsists of essentially two or three different mutant fragments, one ortwo linkers and, optionally, an N-terminal cysteine; and/or apolypeptide which consists of essentially two or three different mutantfragments, an N-terminal extension of the fragment consisting of at most24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 or 11 amino acids,preferably at most 10, 9, 8, 7 or 6 amino acids, still more preferablyat most 5, 4, 3, 2 or 1 amino acid(s), wherein the N-terminal extensionis located directly N-terminally from the fragment in the respectiveBorrelia OspA and, optionally, an N-terminal cysteine. The N-terminalcysteine may optionally be followed by a short peptide linker from 1-10amino acids long, and preferably takes the form of an N-terminal CSSpeptide (SEQ ID NO: 210) or CKQN peptide (SEQ ID NO: 211).

In a second aspect, the present invention relates to a nucleic acidencoding for the polypeptide according to the first aspect.

Nucleic acid molecule as used herein generally refers to any ribonucleicacid molecule or deoxyribonucleic acid molecule, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, nucleic acidmolecule as used herein refers to at least single- and double-strandedDNA, hybrid molecules comprising DNA and RNA that may be single-strandedor, more typically, double-stranded, or a mixture of single- anddouble-stranded regions. As used herein, the term nucleic acid moleculeincludes DNA or RNA molecules as described above that contain one ormore modified bases. Thus, DNA or RNA molecules with backbones modifiedfor stability or for other reasons are “nucleic acid molecule” as thatterm is intended herein. Moreover, DNA or RNA species comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are also nucleic acid molecules as definedherein. It will be appreciated that a great variety of modificationshave been made to DNA and RNA molecules that serve many useful purposesknown to those of skill in the art. The term nucleic acid molecule asused herein embraces such chemically, enzymatically or metabolicallymodified forms of nucleic acid molecules, as well as the chemical formsof DNA and RNA characteristic of viruses and cells, including simple andcomplex cells, inter alia. The term nucleic acid molecule alsoencompasses short nucleic acid molecules often referred to asoligonucleotide(s). The terms “polynucleotide” and “nucleic acid” or“nucleic acid molecule” are used interchangeably herein.

The nucleic acids according to the present invention may be chemicallysynthesized. Alternatively, the nucleic acids can be isolated fromBorrelia and modified by methods known to one skilled in the art. Thesame applies to the polypeptides according to the present invention.

Furthermore, the nucleic acid of the present invention can befunctionally linked, using standard techniques such as cloning, to anydesired sequence(s), whether a Borrelia regulatory sequence or aheterologous regulatory sequence, heterologous leader sequence,heterologous marker sequence or a heterologous coding sequence to createa fusion gene.

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA or cRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or produced bychemical synthesis techniques or by a combination thereof. The DNA maybe triple-stranded, double-stranded or single-stranded. Single-strandedDNA may be the coding strand, also known as the sense strand, or it maybe the non-coding strand, also referred to as the anti-sense strand.

The nucleic acid of the present invention may be comprised in a vectoror in a cell. The vector may comprise the above-mentioned nucleic acidin such a manner that the vector is replicable and can express theprotein encoded by the nucleotide sequence in a host cell.

A great variety of expression vectors can be used to express thepolypeptides according to the present invention. Generally, any vectorsuitable to maintain, propagate or express nucleic acids to express apolypeptide in a host may be used for expression in this regard. Inaccordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single- or double-stranded phage vector ora single- or double-stranded RNA or DNA viral vector. Starting plasmidsdisclosed herein are either commercially available, publicly available,or can be constructed from available plasmids by routine application ofwell-known, published procedures. Preferred among vectors, in certainrespects, are those for expression of nucleic acid molecules and thepolypeptides according to the present invention. Nucleic acid constructsin host cells can be used in a conventional manner to produce the geneproduct encoded by the recombinant sequence. Alternatively, thepolypeptides according to the present invention can be syntheticallyproduced by conventional peptide synthesizers.

In addition, the present invention relates to a host cell comprisingthis vector. Representative examples of appropriate host cells includebacteria, such as streptococci, staphylococci, E. coli, Streptomyces andBacillus subtilis; fungi, such as yeast and Aspergillus; insect cellssuch as Drosophila S2 and Spodoptera Sf9 cells; mammalian cells such asCHO, COS, HeLa, C127, 3T3, BHK, 293 or Bowes melanoma cells; and plantcells. Cell-free translation systems can also be employed to producesuch proteins using RNA derived from the DNA construct of the presentinvention.

In order to express the desired amino acid sequence practically byintroducing the vector according to the present invention into a hostcell, the vector may contain, in addition to the nucleic acid sequenceaccording to the present invention, other sequences for controlling theexpression (e.g., promoter sequences, terminator sequences and enhancersequences) and gene markers for selecting microorganisms, insect cells,animal culture cells, or the like (e.g., neomycin resistance genes andkanamycin resistance genes). Furthermore, the vector may contain thenucleic acid sequence according to the present invention in a repeatedform (e.g., in tandem). The vector may be constructed based onprocedures and manners which are conventionally used in the field ofgenetic engineering.

The host cells may be cultured in an appropriate medium, and the proteinaccording to the present invention may be obtained from the cultureproduct. The protein according to the present invention may be recoveredfrom the culture medium and purified in the conventional manner.

In a third aspect the present invention relates to a pharmaceuticalcomposition comprising the polypeptide according to the first aspectand/or the nucleic acid according to the second aspect and, optionally,a pharmaceutically acceptable carrier or excipient. Preferably, thepharmaceutical composition is used as a medicament, particularly as avaccine or for preventing or treating an infection caused by Borreliaspecies, more preferably pathogenic Borrelia species as disclosed hereinmore preferably comprising B. burgdorferi s.s., B. afzelii, B.bavariensis and B. garinii, and/or other pathogens against which theantigens have been included in the vaccine.

The pharmaceutical composition may contain any pharmaceuticallyacceptable carrier or excipient, such as buffer substances, stabilisersor further active ingredients, especially ingredients known inconnection with pharmaceutical compositions and/or vaccine production.

The invention also includes immunogenic compositions. In some aspects,an immunogenic composition of the invention comprises any of thecompositions discussed herein and a pharmaceutically acceptable carrier.In various aspects, the immunogenic composition has the property ofinducing production of an antibody that specifically binds an outersurface protein A (OspA) protein. In certain aspects, the immunogeniccomposition has the property of inducing production of an antibody thatspecifically binds Borrelia. In particular aspects, the immunogeniccomposition has the property of inducing production of an antibody thatneutralizes Borrelia. In some aspects, the antibody is produced by ananimal. In further aspects, the animal is a mammal. In even furtheraspects, the mammal is human.

The invention further includes vaccine compositions. In some aspects, avaccine composition of the invention comprises any immunogeniccomposition discussed herein and a pharmaceutically acceptable carrier.In various aspects, the invention includes a combination vaccine. Incertain aspects, a combination vaccine of the invention comprises anyvaccine composition discussed herein in combination with at least asecond vaccine composition. In some aspects, the second vaccinecomposition protects against a tick-borne disease. In various aspects,the tick-borne disease is Rocky Mountain Spotted Fever, Babesiosis,Relapsing Fever, Colorado tick fever, Human monocytic ehrlichiosis(HME), Human granulocytic ehrlichiosis (HGE), Southern Tick-AssociatedRash Illness (STARI), Tularemia, Tick paralysis, Powassan encephalitis,Q fever, Crimean-Congo hemorrhagic fever, Cytauxzoonosis, boutonneusefever, or tick-borne encephalitis. In other aspects, the second vaccinecomposition is a vaccine selected from the group consisting of atick-borne encephalitis vaccine, a Japanese encephalitis vaccine, and aRocky Mountain Spotted Fever vaccine. In various aspects, the secondvaccine composition has a seasonal immunization schedule compatible withimmunization against Borrelia infection or Lyme disease.

The invention also includes methods for inducing an immunologicalresponse in a subject. In various aspects, such methods comprise thestep of administering any of the immunogenic compositions or vaccinecompositions discussed herein to the subject in an amount effective toinduce an immunological response. In certain aspects, the immunologicalresponse comprises production of an anti-OspA antibody.

The invention includes methods for preventing or treating a Borreliainfection or Lyme disease in a subject. In various aspects, such methodscomprise the step of administering any of the vaccine compositionsdiscussed herein or any of the combination vaccines discussed herein tothe subject in an amount effective to prevent or treat the Borreliainfection or Lyme disease.

The invention includes uses of compositions of the invention for thepreparation of medicaments. Other related aspects are also provided inthe instant invention.

A preferable carrier or excipient for the polypeptides according to thepresent invention in their diverse embodiments, or a nucleic acidmolecule according to the present invention is an immunostimulatorycompound such as an adjuvant for further stimulating the immune responseto the polypeptide according to the present invention or a codingnucleic acid molecule thereof.

Adjuvants which may be used in compositions of the invention include,but are not limited to:

A. Mineral-Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminium salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.,oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates),sulphates, etc., or mixtures of different mineral compounds, with thecompounds taking any suitable form (e.g., gel, crystalline, amorphous,etc.), and with adsorption being preferred. The mineral containingcompositions may also be formulated as a particle of metal salt.

A useful aluminium phosphate adjuvant is amorphous aluminiumhydroxyphosphate with PO₄/Al molar ratio between 0.84 and 0.92. Anotheruseful aluminium-based adjuvant is AS04, a combination of aluminiumhydroxide+monophosphoryl lipid A (MPL).

B. Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-in-water emulsions, such as MF59 (5% Squalene, 0.5%Tween 80, and 0.5% Span 85, formulated into submicron particles using amicrofluidizer), AS03 (squalene, DL-α-tocopherol and Tween 80) and AF03(squalene, Montane® 80 and Eumulgon® B1 PH). Complete Freund's adjuvant(CFA) and incomplete Freund's adjuvant (IFA) may also be used.

Useful oil-in-water emulsions typically include at least one oil and atleast one surfactant, with the oil(s) and surfactant(s) beingbiodegradable (metabolizable) and biocompatible. The oil droplets in theemulsion are generally less than 1 μm in diameter, with these smallsizes being achieved with a microfluidizer to provide stable emulsions.Droplets with a size less than 220 nm are preferred as they can besubjected to filter sterilization.

The emulsion can comprise oils such as those from an animal (such asfish) or vegetable source. Sources for vegetable oils include nuts,seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil,the most commonly available, exemplify the nut oils. Jojoba oil can beused e.g., obtained from the jojoba bean. Seed oils include saffloweroil, cottonseed oil, sunflower seed oil, sesame seed oil and the like.In the grain group, corn oil is the most readily available, but the oilof other cereal grains such as wheat, oats, rye, rice, teff, triticaleand the like may also be used. 6-10 carbon fatty acid esters of glyceroland 1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and may therefore be used in thepractice of this invention. The procedures for separation, purification,saponification and other means necessary for obtaining pure oils fromanimal sources are well known in the art. Most fish containmetabolizable oils which may be readily recovered. For example, codliver oil, shark liver oils, and whale oil such as spermaceti exemplifyseveral of the fish oils which may be used herein. A number of branchedchain oils are synthesized biochemically in 5-carbon isoprene units andare generally referred to as terpenoids. Shark liver oil contains abranched, unsaturated terpenoid known as squalene,2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which isparticularly preferred herein. Squalane, the saturated analog tosqualene, is also a preferred oil. Fish oils, including squalene andsqualane, are readily available from commercial sources or may beobtained by methods known in the art. Other preferred oils are thetocopherols (see below). Mixtures of oils can be used.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophilebalance). Preferred surfactants of the invention have a HLB of at least10, preferably at least 15, and more preferably at least 16. Theinvention can be used with surfactants including, but not limited to:the polyoxyethylene sorbitan esters surfactants (commonly referred to asthe Tweens), especially polysorbate 20 and polysorbate 80; copolymers ofethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO),sold under the DOWF AX™ tradename, such as linear EO/PO blockcopolymers; octoxynols, which can vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy) polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe Tergitol™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters (commonly known as the SPANs), such as sorbitan trioleate (Span85) and sorbitan monolaurate. Non-ionic surfactants are preferred.Preferred surfactants for including in the emulsion are Tween 80(polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate),lecithin and Triton X-100.

Mixtures of surfactants can be used e.g., Tween 80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (Tween 80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1%;octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or otherdetergents in the Triton series) 0.001 to 0.1%, in particular 0.005 to0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Preferably, substantially all (e.g. at least 90% by number) of the oildroplets have a diameter of less than 1 μm, e.g. <750 nm, <500 nm, <400nm, <300 nm, <250 nm, <220 nm, <200 nm, or smaller. One specific usefulsubmicron emulsion consists of squalene, Tween 80, and Span 85. Thecomposition of the emulsion by volume can be about 5% squalene, about0.5% polysorbate 80 and about 0.5% Span 85. In weight terms, theseratios become 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85. TheMF59 emulsion advantageously includes citrate ions e.g. 10 mM sodiumcitrate buffer.

C. Saponin Formulations

Saponin formulations may also be used as adjuvants in the invention.Saponins are a heterogeneous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponin from the bark of theQuillaia saponaria Molina tree has been widely studied as adjuvant.Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brideal veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs. QS21 is marketed as Stimulon™.

Saponin compositions have been purified using HPLC and RP-HPLC. Specificpurified fractions using these techniques have been identified,including QS7, QS 17, QS 18, QS21, QH-A, QH-B and QH-C. Preferably, thesaponin is QS21. Saponin formulations may also comprise a sterol, suchas cholesterol.

Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexes (ISCOMs). ISCOMs typicallyalso include a phospholipid such as phosphatidylethanolamine orphosphatidylcholine. Any known saponin can be used in ISCOMs.Preferably, the ISCOM includes one or more of QS7, QS 17, QS 18, QS21,QH-A, QH-B and QH-C. Optionally, the ISCOMS may be devoid of additionaldetergent.

D. Virosomes and Virus-Like Particles

Virosomes and virus-like particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retroviruses, Norwalk virus, Human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein pi).

E. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as non-toxic derivatives of enterobacteriallipopolysaccharide (LPS), Lipid A derivatives, immunostimulatoryoligonucleotides and ADP-ribosylating toxins and detoxified derivativesthereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. Such “smallparticles” of 3dMPL are small enough to be sterile filtered through a0.22 μm membrane. Other non-toxic LPS derivatives include monophosphoryllipid A mimics, such as aminoalkyl glucosaminide phosphate derivativese.g. RC-529 and the synthetic phospholipid dimer, E6020.

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. Immunostimulatory oligonucleotides suitable for use asadjuvants in the invention include nucleotide sequences containing a CpGmotif (a dinucleotide sequence containing an unmethylated cytosinelinked by a phosphate bond to a guanosine). Double-stranded RNAs andoligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. The CpG sequence may be directed to TLR9, such as themotif GTCGTT or TTCGTT. The CpG sequence may be specific for inducing aTh1 immune response, such as a CpG-A ODN, or it may be more specific forinducing a B cell response, such a CpG-B ODN. Preferably, the CpG is aCpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. Aparticularly useful adjuvant based around immunostimulatoryoligonucleotides is known as IC31®. Thus an adjuvant used with theinvention may comprise a mixture of (i) an oligonucleotide {e.g. between15-40 nucleotides) including at least one (and preferably multiple) CpImotifs (i.e. a cytosine linked to an inosine to form a dinucleotide),and (ii) a polycationic polymer, such as an oligopeptide (e.g. between5-20 amino acids) including at least one (and preferably multiple)Lys-Arg-Lys tripeptide sequence(s). The oligonucleotide may be adeoxynucleotide comprising the 26-mer sequence 5′-(dIdC)₁₃-3′ (SEQ IDNO: 32). The polycationic polymer may be a peptide comprising the 11-meramino acid sequence KLKLLLLLKLK (SEQ ID NO: 33).

Polycationic compounds derived from natural sources include HIV-REV orHIV-TAT (derived cationic peptides, antennapedia peptides, chitosan orother derivatives of chitin) or other peptides derived from thesepeptides or proteins by biochemical or recombinant production. Otherpreferred polycationic compounds are cathelin or related or derivedsubstances from cathelin. For example, mouse cathelin is a peptide,which has the amino acid sequenceNH₂-RLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE-COOH (SEQ ID NO: 31). Relatedor derived cathelin substances contain the whole or parts of thecathelin sequence with at least 15-20 amino acid residues. Derivationsmay include the substitution or modification of the natural amino acidsby amino acids which are not among the 20 standard amino acids.Moreover, further cationic residues may be introduced into such cathelinmolecules. These cathelin molecules are preferred to be combined withthe antigen. These cathelin molecules surprisingly have turned out to bealso effective as an adjuvant for an antigen without the addition offurther adjuvants. It is therefore possible to use such cathelinmolecules as efficient adjuvants in vaccine formulations with or withoutfurther immune activating substances.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (E. coli heat labile enterotoxin “LT”), Vibriocholerae (Cholera toxin “CT”), or Bordetella pertussis (Pertussis toxin“PT”). The use of detoxified ADP-ribosylating toxins as mucosaladjuvants and as parenteral adjuvants is known. The toxin or toxoid ispreferably in the form of a holotoxin, comprising both A and B subunits.Preferably, the A subunit contains a detoxifying mutation; preferablythe B subunit is not mutated. Preferably, the adjuvant is a detoxifiedLT mutant such as LT-K63, LT-R72, LT-G192 or dmLT. A useful CT mutant isCT-E29H.

F. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophagecolony stimulating factor and tumor necrosis factor. A preferredimmunomodulator is IL-12.

G. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres or mucoadhesives such as cross-linked derivatives ofpolyacrylic acid, polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention.

H. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g., a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, apoly(lactide-co-glycolide) etc.), wherein poly(lactide-co-glycolide) arepreferred, optionally treated to have a negatively-charged surface (e.g.with SDS) or a positively-charged surface (e.g., with a cationicdetergent, such as CTAB).

I. Liposomes

Examples of liposome formulations suitable for use as adjuvants areknown.

J. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters. Such formulations further includepolyoxyethylene sorbitan ester surfactants in combination with anoctoxynol as well as polyoxyethylene alkyl ethers or ester surfactantsin combination with at least one additional non-ionic surfactant such asan octoxynol. Preferred polyoxyethylene ethers are selected from thefollowing group: polyoxyethylene-9-lauryl ether (laureth 9),polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether.

K. Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-5n-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

L. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use as adjuvants inthe invention include Imiquimod and its homologues (e.g., “Resiquimod3M”).

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above.

Preferably, the immunostimulatory compound in the pharmaceuticalpreparation according to the present invention is selected from thegroup of polycationic substances, especially polycationic peptides,immunostimulatory nucleic acids molecules, preferably immunostimulatorydeoxynucleotides, oil-in-water or water-in-oil emulsions, MF59, alum,alum salts, Freund's complete adjuvant, Freund's incomplete adjuvant,neuroactive compounds, especially human growth hormone, or combinationsthereof.

The use of an aluminium hydroxide and/or aluminium phosphate adjuvant isparticularly preferred, and antigens are generally adsorbed to thesesalts.

Also, the pharmaceutical composition in accordance with the presentinvention is a pharmaceutical composition which comprises at least anyof the following compounds or combinations thereof: the nucleic acidmolecules according to the present invention, the polypeptides accordingto the present invention in their diverse embodiments, the vectoraccording to the present invention, the cells according to the presentinvention, the antibody according to the present invention, thefunctional nucleic acids according to the present invention and thebinding peptides such as the anticalines and high-affinity bindingpeptides and peptide aptamers, respectively, according to the presentinvention, any agonists and antagonists according to the presentinvention, preferably screened as described herein. In connectiontherewith, any of these compounds may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such carriers may include, but are notlimited to, saline, buffered saline, dextrose, water, glycerol, ethanoland combinations thereof. The formulation should suit the mode ofadministration.

The pharmaceutical compositions of the present invention may beadministered in any effective, convenient manner including, forinstance, administration by topical, oral, anal, vaginal, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal, intratrachealor intradermal routes, among others.

In therapy or as a prophylactic, the active agent of the pharmaceuticalcomposition of the present invention may be administered to anindividual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition, preferably the pharmaceutical compositionmay be formulated for topical application, for example in the form ofointments, creams, lotions, eye ointments, eye drops, ear drops,mouthwash, impregnated dressings and sutures and aerosols, and maycontain appropriate conventional additives, including, for example,preservatives, solvents to assist drug penetration, and emollients inointments and creams. Such topical formulations may also containcompatible conventional carriers, for example cream or ointment bases,and ethanol or oleyl alcohol for lotions. Such carriers may constitutefrom about 1% to about 98% by weight of the formulation; more usuallythey will constitute up to about 80% by weight of the formulation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

In a preferred embodiment the pharmaceutical composition is a vaccinecomposition. Preferably, such vaccine composition is conveniently ininjectable form. Conventional adjuvants may be employed to enhance theimmune response. A suitable unit dose for vaccination with a proteinantigen is for adults between 0.02 μg and 3 μg antigen per kg bodyweight and for children between 0.2 μg and 10 μg antigen per kg bodyweight, and such dose is preferably administered 1 to 3 times atintervals of 2 to 24 weeks.

At the indicated dose range, no adverse toxicological effects areexpected with the compounds of the invention, which would preclude theiradministration to suitable individuals.

The pharmaceutical composition can contain a range of differentantigens. Examples of antigens are whole-killed or attenuated organisms,subfractions of these organisms, proteins, or, in their most simpleform, peptides. Antigens can also be recognized by the immune system inthe form of glycosylated proteins or peptides and may also be or containpolysaccharides or lipids. Short peptides can be used, since cytotoxicT-cells (CTL) recognize antigens in the form of short, usually 8-11amino acids long, peptides in conjunction with major histocompatibilitycomplex (MHC). B cells can recognize linear epitopes as short as 4 to 5amino acids, as well as three-dimensional structures (conformationalepitopes).

In a preferred embodiment, the pharmaceutical composition of the thirdaspect additionally comprises a hyperimmune serum-reactive antigenagainst a Borrelia protein or an active fragment or variant thereof,such as, e.g., the antigens, fragments and variants as described in WO2008/031133.

According to the invention, the pharmaceutical composition according tothe third aspect may be used as a medicament, particularly as a vaccine,particularly in connection with particularly a disease or diseasedcondition which is caused by, linked or associated with Borrelia.

Borrelia belongs to the family Spirochaetaceae, which is subdivided intothe medically important genera Treponema, Leptospira and Borrelia. B.burgdorferi s.l. is a spiral-shaped, vigorously motile gram-negativebacterium, about 10-20 μm long and 0.2-0.5 μm wide, that grows undermicroaerophilic conditions. The spirochetal cell wall consists of acytoplasmic membrane surrounded by peptidoglycan and several flagellaand then by a loosely-associated outer membrane.

Lyme borreliosis generally occurs in stages characterized by differentclinical manifestations, with remissions and exacerbations. Stage 1,early infection, consists of a localized infection of the skin, followedwithin days or weeks by stage 2, disseminated infection, and months toyears later by stage 3, persistent infection. However, the infection isvariable; some patients have only localized infections of the skin,while others display only later manifestations of the illness, such asarthritis. Different clinical syndromes of Lyme borreliosis are alsocaused by infection with diverse B. burgdorferi s.l. species. B.burgdorferi s.s. more often causes joint manifestations (arthritis) andheart problems, B. afzelii causes mainly dermal symptoms (erythemamigrans; EM and acrodermatitis chronica atrophicans; ACA), whereas B.garinii is implicated in most cases of neuroborreliosis.

Localized infection—The most common symptom of stage 1 of an infectionis erythema migrans, which occurs in 70-80% of infected people. Thisskin lesion is often followed by flu-like symptoms, such as myalgia,arthralgia, headache and fever. These non-specific symptoms occur in 50%of patients with erythema migrans.

Disseminated infection—During stage 2, the bacteria move into the bloodstream from the site of infection to distal tissues and organs.Neurological, cardiovascular and arthritic symptoms that occur in thisstage include meningitis, cranial neuropathy and intermittentinflammatory arthritis.

Persistent infection—Stage 3 of the infection is chronic and occurs frommonths to years after the tick bite. The most common symptom in NorthAmerica is rheumatoid arthritis, caused by an infection with B.burgdorferi s.s. Persistent infection of the central nervous system withB. garinii causes more severe neurological symptoms during stage 3, anda persistent infection of the skin with B. afzelii results inacrodermatitis chronica atrophicans.

The pharmaceutical composition of the present invention may be used as amedicament, particularly as a vaccine, particularly in connection with adisease or disease condition which is caused by, linked with orassociated with Borrelia, more preferably any pathogenic Borreliaspecies and more preferably in a method for treating or preventing aBorrelia infection, particularly a B. burgdorferi s.s., B. garinii, B.afzelii, B. andersonii, B. bavariensis, B. bissettii, B. valaisiana, B.lusitaniae, B. spielmanii, B. japonica, B. tanukii, B. turdi or B.sinica infection, preferably a B. burgdorferi s.s., B. afzelii or B.garinii infection.

In connection therewith, it should be noted that the various Borreliaspecies, including B. burgdorferi s.l., comprise several species andstrains including those disclosed herein. A disease related, caused orassociated with the bacterial infection to be prevented and/or treatedaccording to the present invention includes Lyme borreliosis (Lymedisease). Further aspects, symptoms, stages and subgroups of Lymeborreliosis as well as specific groups of patients suffering from suchdisease as also disclosed herein, including in the introductory part,are incorporated herein by reference. More specifically, Lymeborreliosis generally occurs in stages, with remission and exacerbationswith different clinical manifestation at each stage. Early infectionstage 1 consists of localized infection of the skin, followed withindays or weeks by stage 2, disseminated infection, and months to yearslater by stage 3, persistent infection. However, the infection isvariable; some patients have only localized infections of the skin,while others display only later manifestations of the illness, such asarthritis.

In a fourth aspect, the present invention relates to a method oftreating or preventing a Borrelia infection in a subject in needthereof, comprising the step of administering to the subject atherapeutically effective amount of a pharmaceutical compositionaccording to the third aspect.

The term “subject” is used throughout the specification to describe ananimal, preferably a mammal, more preferably a human, to whom atreatment or a method according to the present invention is provided.For treatment of those infections, conditions or disease states whichare specific for a specific animal such as a human patient, the termpatient refers to that specific animal Preferably, the subject is ahuman; however, the medical use of the composition may also includeanimals such as poultry including chicken, turkey, duck or goose,livestock such as horse, cow or sheep, or companion animals such as dogsor cats.

The term “effective amount” is used throughout the specification todescribe an amount of the present pharmaceutical composition which maybe used to induce an intended result when used in the method of thepresent invention. In numerous aspects of the present invention, theterm effective amount is used in conjunction with the treatment orprevention. In other aspects, the term effective amount simply refers toan amount of an agent which produces a result which is seen as beingbeneficial or useful, including in methods according to the presentinvention where the treatment or prevention of a Borrelia infection issought.

The term effective amount with respect to the presently describedcompounds and compositions is used throughout the specification todescribe that amount of the compound according to the present inventionwhich is administered to a mammalian patient, especially including ahuman patient, suffering from a Borrelia-associated disease, to reduceor inhibit a Borrelia infection.

In a preferred embodiment, the method of immunizing a subject accordingto the fourth aspect comprises the step of administering to the subjecta therapeutically effective amount of a pharmaceutical composition ofthe third aspect of the current invention.

The method comprises inducing an immunological response in an individualthrough gene therapy or otherwise, by administering a polypeptide ornucleic acid according to the present invention in vivo in order tostimulate an immunological response to produce antibodies or acell-mediated T cell response, either cytokine-producing T cells orcytotoxic T cells, to protect said individual from disease, whether ornot that disease is already established within the individual.

The products of the present invention, particularly the polypeptides andnucleic acids, are preferably provided in isolated form, and may bepurified to homogeneity. The term “isolated” as used herein meansseparated “by the hand of man” from its natural state; i.e., if itoccurs in nature, it has been changed or removed from its originalenvironment, or both. For example, a naturally-occurring nucleic acidmolecule or a polypeptide naturally present in a living organism in itsnatural state is not “isolated”, but the same nucleic acid molecule orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein. As part of or followingisolation, such nucleic acid molecules can be joined to other nucleicacid molecules, such as DNA molecules, for mutagenesis, to form fusiongenes, and for propagation or expression in a host, for instance. Theisolated nucleic acid molecules, alone or joined to other nucleic acidmolecules such as vectors, can be introduced into host cells, in cultureor in whole organisms. Introduced into host cells in culture or in wholeorganisms, such DNA molecules still would be isolated, as the term isused herein, because they would not be in their naturally-occurring formor environment. Similarly, the nucleic acid molecules and polypeptidesmay occur in a composition, such as medium formulations, solutions forintroduction of nucleic acid molecules or polypeptides, for example,into cells, compositions or solutions for chemical or enzymaticreactions, for instance, which are not naturally occurring compositions,and, therein remain isolated nucleic acid molecules or polypeptideswithin the meaning of that term as it is employed herein.

The invention is not limited to the particular methodology, protocolsand reagents described herein because they may vary. Furthermore, theterminology used herein is for the purpose of describing particularembodiments only and is not intended to limit the scope of the presentinvention. As used herein and in the appended claims, the singular forms“a”, “an”, and the include plural reference unless the context clearlydictates otherwise. Similarly, the words “comprise”, “contain” and“encompass” are to be interpreted inclusively rather than exclusively.

Unless defined otherwise, all technical and scientific terms and anyacronyms used herein have the same meanings as commonly understood byone of ordinary skill in the art in the field of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice of the present invention, thepreferred methods, and materials are described herein.

The present invention is further illustrated by the following Figures,Tables, Examples and the Sequence listing, from which further features,embodiments and advantages may be taken. As such, the specificmodifications discussed are not to be construed as limitations on thescope of the invention. It will be apparent to the person skilled in theart that various equivalents, changes, and modifications may be madewithout departing from the scope of the invention, and it is thus to beunderstood that such equivalent embodiments are to be included herein.

In connection with the present invention

FIGS. 1A-1C schematically shows the production of mutant OspA fragmentheterodimers according to the current invention.

FIG. 2 schematically represents the polypeptide components of onepossible pharmaceutical composition of the current invention comprisingthree different mutant OspA heterodimers.

FIG. 3 shows the chemical structure of Pam₃Cys, an example of a fattyacid substituted cysteine, such as would be found at the N-terminus oflipidated polypeptides of the current invention.

Table 1 shows the thermal stability of the folding of mutant serotype 2OspA fragments with disulfide bond types from D1 to D5 (see Table A-4)compared to the wild-type serotype 2 OspA fragment without disulfidebonds (D0).

Table 2 shows the protection of mice from B. afzelii infection by theTick Challenge Method following immunization with mutant serotype 2 OspAfragments with disulfide bond types D1 to D5, including control groupsof mice immunized with PBS, full-length OspA or the wild-type serotype 2OspA fragment.

Table 3 shows the protection of mice from B. afzelii infection by theTick Challenge Method following immunization with lipidated mutantserotype 2 OspA fragments with disulfide bond types D2, D3 and D4,including control groups of mice immunized with PBS or full-length OspAprotein.

Table 4 shows the protection of mice from B. burgdorferi s.s. deliveredby needle challenge or from B. afzelii delivered by tick challenge byimmunization with lipidated His-tagged mutant OspA serotype 1/serotype 2fragment heterodimer (Lip-S1D1-S2D1-His). Control groups included miceimmunized with lipidated His-tagged mutant OspA serotype 1 (Lip-M1B-His)or serotype 2 (Lip-M2B-His) fragment monomers individually or withadjuvant alone.

The figures and tables which may be referred to in the specification aredescribed below in more detail.

FIGS. 1A-1C Production of a mutant OspA heterodimer of the inventioncomprising mutant OspA C-terminal fragments from two different serotypesof Borrelia sp. (FIG. 1A) Schematic representation of a nucleic acidencoding a lipidated mutant OspA heterodimer. The components, from 5′ to3′ comprise the coding sequences for a lipidation signal sequence (Lipsignal), a small cysteine-containing peptide for N-terminal lipidation(Lipidation peptide=LP), a mutant C-terminal fragment of OspA with twonon-native cysteines, a short linker peptide, LN1, followed by a secondmutant OspA C-terminal fragment with two non-native cysteines. (FIG. 1B)The intermediate mutant OspA heterodimer polypeptide comprises thenascent product directly following translation of the nucleic acidconstruct. From the N- to the C-terminus, this polypeptide consists of alipidation signal sequence (Lip signal), a cysteine-containing peptidefor lipidation (LP), a mutant OspA fragment with a non-native disulfidebond, a short linker peptide, LN1, followed by a second mutant OspAfragment with a non-native disulfide bond. (FIG. 1C) The final lipidatedmutant OspA heterodimer polypeptide after post-translationalmodification. The heterodimer, from the N- to the C-terminus, consistsof a short cysteine-containing peptide with the N-terminal cysteinelipidated (indicated by “Lip”), a mutant OspA fragment stabilized by adisulfide bond, a linker peptide, LN1, and a second mutant OspA fragmentstabilized by a disulfide bond. The lipidation signal sequence iscleaved off during post-translational modification of the polypeptide asshown.

FIG. 2 An example of a preferred pharmaceutical composition according tothe current invention. Three mutant OspA heterodimers, each comprisingmutated OspA fragments from two different Borrelia serotypes are presentin the composition, together providing OspA antigens from six differentBorrelia serotypes. Such a pharmaceutical composition enablessimultaneous immunization against six of the most prevalent serotypes ofBorrelia.

FIG. 3 Diagram of the chemical structure of Pam₃Cys, an example of afatty acid substitution of the N-terminal cysteine of full-lengthwild-type OspA protein as well as of lipidated mutant OspA fragmentmonomers and heterodimers of the invention. During post-translationalmodification of a full-length OspA protein or polypeptides of theinvention, the N-terminal lipidation signal sequence is cleaved off andfatty acids, most commonly three palmitoyl moieties (“Pam₃”), areenzymatically covalently attached to the N-terminal cysteine residue(the S atom of which is indicated by an arrow). The remaining residuesof the polypeptide chain, which are located C-terminally from thePam₃Cys residue, are represented by “Xn”. (Modified from Bouchon, et al.(1997) Analytical Biochemistry 246: 52-61.)

TABLE 1 Thermal stability of non-lipidated, His-tagged B. afzelii K78mutant serotype 2 OspA fragments with different placement of disulfidebonds. Mutant serotype 2 OspA fragments with different cysteine bondtypes (see Table A-4) were solubilized in 50 mM Tris-HCl, 150 mM NaCl(pH 8.0) and tested for thermal stability compared with the wild-typeserotype 2 OspA fragment (S2D0). The presence of a disulfide bondresulted in an increased melting temperature compared to the wild-typeserotype 2 OspA fragment. Serotype 2 OspA mutant Melting temperaturefragment SEQ ID NO: (° C.) S2D0-His* 1 47.6 S2D1-His 2 70.4 S2D2-His 354.6 S2D3-His 4 58.6 S2D4-His 5 58.4 S2D5-His 6 53.8 *see Tables A-4 andA-5 for nomenclature.

TABLE 2 Protection of mice from B. afzelii infection by the tickchallenge method by immunization with non-lipidated mutant serotype 2OspA fragments. Five non-lipidated mutant serotype 2 OspA fragments weretested for protective capacity at two different doses (30 μg and 5 μg)and compared with the wild-type serotype 2 fragment. Groups of miceimmunized with adjuvant alone or with non-lipidated full-length serotype2 OspA served as negative and positive controls, respectively. Allantigens were His-tagged and non-lipidated. The data presented combinethe results of several experiments performed under identical conditions.3 × 30 μg 3 × 5 μg (data from 11 experiments) (data from 4 experiments)(all groups include Al(OH)₃) (all groups include Al(OH)₃) InfectedInfected Immunogen mice total mice p-value mice total mice p-valueAdjuvant alone 67 73 n/a 20 23 n/a Full-length OspA K78- 15 87 <0.001***0 25 <0.001*** His (SEQ ID NO: 209) S2D0-His 20 27 0.045* 5 16 0.001***(SEQ ID NO: 1) S2D1-His 7 32 <0.001*** 1 25 <0.001*** (SEQ ID NO: 2)S2D2-His 2 29 <0.001*** 3 26 <0.001*** (SEQ ID NO: 3) S2D3-His 10 44<0.001*** 0 21 <0.001*** (SEQ ID NO: 4) S2D4-His 6 35 <0.001*** 3 27<0.001*** (SEQ ID NO: 5) S2D5-His 6 37 <0.001*** 2 11 <0.001*** (SEQ IDNO: 6) *significant (≦0.05), ** highly significant (≦0.01), ***extremelysignificant (≦0.001), Fisher's exact test, two-tailed.

TABLE 3 Protection of mice from B. afzelii infection by the TickChallenge Method by immunization with decreasing doses of lipidatedmutant serotype 2 OspA fragments. Three lipidated mutant serotype 2 OspAfragments with different disulfide bond types were tested for protectivecapacity. Groups of mice immunized with adjuvant alone or with non-lipidated full-length serotype 2 OspA served as negative and positivecontrols, respectively. All antigens were His-tagged. 3 × 3 μg 3 × 1 μg3 × 0.3 μg (data from 3 experiments) (data from 5 experiments) (datafrom 4 experiments) (all groups included Al(OH)₃) (all groups includedAl(OH)₃) (all groups included Al(OH)₃) Infected Infected InfectedImmunogen mice Total mice p-value mice Total mice p-value mice Totalmice p-value Adjuvant 22 24 n/a 33 37 n/a 28  30 n/a alone Full-length 014 <0.001*** 0 21 <0.001*** n/a n/a n/a OspA K78- His (SEQ ID NO: 209)Lip-S2D2- 0 17 <0.001*** 0 15 <0.001*** 0 21 <0.001*** His (SEQ ID NO:142) Lip-S2D3- 1 15 <0.001*** 1 12 <0.001*** 5 19 <0.001*** His (SEQ IDNO: 143) Lip-S2D4- 0 8 <0.001*** 0 14 <0.001*** 0 19 <0.001*** His (SEQID NO: 144) * significant (<0.05), ** highly significant (<0.01),***extremely significant (≦0.001), Fisher's exact test, two-tailed.

TABLE 4 Protection of mice from Borrelia infection by both needlechallenge (B. burgdorferi s.s.) and Tick Challenge Method (B. afzelii)by immunization with lapidated His-tagged mutant serotype1/serotype 2OspA fragment heterodimers. The lipidated His-tagged mutant serotype1/serotype 2 OspA fragment heterodimer protein (Lip-S1D1-S2D1-His) wastested for protective capacity. Groups of mice immunized three times attwo week intervals with adjuvant alone or with lipidated His-taggedmutant serotype 1 (Lip-S1D1-His) or serotype 2 (Lip-S2D1-His) OspAfragment monomers individually served as negative and positive controls,respectively. Immunized mice were challenged two weeks after the lastimmunization with either B. burgdorferi s.s. via needle challenge(Experiments 1-3) or with B. afzelii via infected ticks (Experiments4-6). All antigens were lipidated and His-tagged. Needle challengeInfected/ Infected/ Infected/ (serotype 1: Total Total Total ImmunogenDose B. burgdorferi s.s.) Exp. 1 Exp. 2 Exp. 3 Lip-S1D1-S2D1- 3 × 5.0N40 (ST1) 0/10***  0/9***   4/10**

Lip-S1D1-His 3 × 2.5 N40 (ST1) 2/10***  1/10*   4/10** Lip-S2D1-His 3 ×2.5 N40 (ST1) 1/10*** 3/10  5/10* Adjuvant alone — N40 (ST1) 10/10  8/10 10/10 Tick challenge (Serotype 2: B. afzelii) Exp. 4 Exp. 5 Exp. 6Lip-S1D1-S2D1- 3 × 2.0 Tick (ST2) 0/10***  0/9***   0/6***

Lip-S1D1-His 3 × 1.0 Tick (ST2) 2/10***  2/8** 2/4 Lip-S2D1-His 3 × 1.0Tick (ST2) 1/8***   0/4**  0/4** Adjuvant alone — Tick (ST2) 9/9   8/8 7/7 P-value; Fisher's exact test, two tailed. *significant (<0.05),**highly significant (<0.01), ***extremely significant (<0.001)

indicates data missing or illegible when filed

Examples Example 1. Assessment of Thermal Stability of Mutant Serotype 2OspA Fragments

Experimental Procedures

Thermal Stability

The melting temperatures (T_(m)) of non-lipidated mutant serotype 2 OspAfragment monomers were determined by the fluorescence-based thermalshift assay described by Pantoliano, et al. (J. Biomol Screen 6:429-440(2001)). The fluorescent dye SYPRO® Orange protein gel stain (suppliedas a 5000× concentrate in DMSO by Sigma, U.S.A) was used to monitorprotein unfolding. In each well, 7.5 μl of SYPRO® Orange (diluted 1:1000from the stock solution) and 17.5 μl of a solution of protein (1 μg or 2μg) in buffer were combined. The protein samples were heated from 25° C.to 95° C. at a rate of 0.2° C./10 sec in the CFX96 Real-time DetectionSystem (Bio-Rad, USA) and fluorescent changes were monitored.Fluorescence intensity was measured with excitation and emissionwavelengths of 490 and 575 nm, respectively. The Tm was determined usingthe Bio-Rad CFX Manager 2.0 program. The Tm values of non-lipidatedHis-tagged serotype 2 OspA mutant fragments were measured in fourdifferent buffer systems: 50 mM Tris-HCl, 150 mM NaCl (pH 9.0); 50 mMTris-HCl, 150 mM NaCl (pH 8.0); PBS (pH 7.4); and 25 mM HEPES, 150 mMNaCl (pH 6.5), using the non-lipidated serotype 2 OspA wild-typefragment (52D0) as a control.

Results

In all cases, mutant serotype 2 OspA fragments with an introducedcysteine bond had higher melting temperatures than the wild-typeserotype 2 OspA fragment (S2D0) (see Table 1). The melting temperatureswere tested in four different buffer systems with similar results (datafor proteins dissolved in 50 mM Tris-HCl, 150 mM NaCl (pH 8.0) is shownin Table 1), indicating that the stability of the proteins is similarover a wide pH range. This result lends credence to the hypothesis thatthe introduced disulfide bond stabilizes the OspA fragment.

Example 2. Protection of Mice from Infection with B. afzelii in the TickChallenge Method by Immunization with Non-Lipidated his-Tagged MutantSerotype 2 OspA Fragment Monomers

Experimental Procedures

Cloning and Expression of Recombinant Proteins

The wild-type serotype 2 OspA fragment as well as the serotype 2 mutantOspA fragments with cysteine bond types 1-5 (SEQ ID NOs: 1, 2, 3, 4, 5and 6, respectively), were codon-optimized for E. coli expression byGenScript, USA. The non-lipidated serotype 2 mutant OspA fragments wereC-terminally histidine-tagged for purification purposes. Gene fragmentswere cloned into the pET28b(+) vector (Novagen, USA), a vectorcontaining a Kanamycin resistance cassette as well as a T7 promoter. Themonomers were expressed in BL21 Star™(DE3) cells (Invitrogen, USA) at37° C. by the addition of IPTG. Cells were collected after 4 h bycentrifugation and the pellet was stored at −70° C. for up to 12 monthsprior to further processing.

Purification of Non-Lipidated his-Tagged Wild-Type and Mutant OspAFragment Monomer Proteins

Cells were disrupted mechanically by high-pressure homogenization andthe soluble fraction containing the His-tagged OspA fragments wasapplied to a Ni-sepharose column (Ni Sepharose™ 6 Fast Flow; GEHealthcare, United Kingdom) and the His-tagged OspA fragments wereeluted on an Imidazole gradient (0-250 mM). Pooled fractions werefurther purified over a gel filtration column (Superdex 200, GEHealthcare) followed by a buffer exchange column (Sephadex G-25, GEHealthcare). His-tagged OspA fragment peaks were pooled on the basis ofthe analytical size exclusion column and reversed phase chromatography.After sterile filtration, the purified proteins were stored at −20° C.until formulation.

Immunization of Mice

Female C3H/HeN (H-2^(k)) mice were used for all studies (Harlan, Italy).Prior to each challenge, groups of five 8-week-old mice were bled viathe tail vein and pre-immune sera were prepared and pooled. Fivenon-lipidated mutant serotype 2 OspA fragment proteins (S2D1-5, SEQ IDNOs: 2, 3, 4, 5 and 6, respectively), were tested in fifteen separateexperiments. Three subcutaneous (s c) immunizations of 100 μL, wereadministered at two week intervals. Doses used were 30 and 5 μg of therespective protein, tested in 11 and 4 experiments respectively. Allformulations included aluminium hydroxide (Al(OH)₃) at a finalconcentration of 0.15%. One week after the third immunization, blood wascollected and hyper-immune sera were prepared. In each experiment, onegroup injected with PBS formulated with Al(OH)₃ was included as anegative control and one group of mice was immunized with S2D0, thewild-type C-terminal OspA fragment from B. afzelii strain K78 (SEQ IDNO: 1). Another group immunized with a non-lipidated full-lengthwild-type OspA protein from B. afzelii, strain K78 (SEQ ID NO: 209),also formulated with 0.15% Al(OH)₃, was included as positive control ineach animal study. All animal experiments were conducted in accordancewith Austrian law (BGB1 Nr. 501/1989) and approved by“Magistratsabteilung 58”.

Tick Challenge of Immunized Mice and Collection of Sera and Tissues(Herein Referred to Also as “Tick Challenge Method”)

Tick challenge of immunized mice was done two weeks after the lastimmunization. In order to challenge the immunized mice with B. afzelii,the hair of the back of each mouse was removed with Veet® Cream (ReckittBenckiser, United Kingdom) and a small ventilated container was glued tothe skin with super glue (Pattex, Germany). Thereafter, one or two I.ricinus nymphs infected with B. afzelii, strain IS1, were applied permouse, allowed to attach and feed to depletion. The feeding status wasmonitored for each individual tick and only mice where at least onefully-fed tick was collected were included in the final readout. Nodistinction was made between mice where one or two fully-fed ticks werecollected.

Six weeks after the tick application, blood was collected by orbitalbleeding and final sera were prepared and used for VlsE ELISA analysisto determine infection status. The mice were then sacrificed by cervicaldislocation and one ear from each mouse was collected, DNA extracted andsubjected to nested PCR analysis to identify Borrelia in tissue.

Infection Readout

Only mice where the applied tick(s) fed to completion and could becollected were included in the final readout of the experiment. The micewere sacrificed 6 weeks after tick application and organs as well asfinal sera were collected. The final infection readout was based on twodifferent analyses (nested PCR targeting the 16S-23S intergenic spacerand VlsE (IR6) ELISA as described in detail below).

Nested PCR Targeting the 16S-23S Intergenic Spacer

One ear from each mouse was subjected to DNA extraction and purificationusing the DNeasy Blood and Tissue Kit (Qiagen, Germany) according to themanufacturer's instructions, with the following modification. Each earwas digested over night at 60° C. in recombinant Proteinase K, PCR grade(Roche, 14-22 mg/mL). The DNA was eluted in 50 μL deionized sterilewater and stored at −20° C. until further analysis. As a negativecontrol, one empty purification column was included in each DNAextraction and purification and the eluate subjected to nested PCR. AllDNA extracts were screened for the presence of Borrelia DNA by a nestedPCR procedure, comprising 40 cycles of 94° C. for 30 s, 56° C. for 30 sand 72° C. for 60 s using the primers; Forward5′-GTATGTTTAGTGAGGGGGGTG-3′ (SEQ ID NO: 26) and Reverse5′-GGATCATAGCTCAGGTGGTTAG-3′ (SEQ ID NO: 27). From the reaction volumeof 10 μL, 1 μL was used as template for the nested PCR reaction. Thenested PCR step comprised 25 cycles of 94° C. for 30 s, 60° C. for 30 sand 72° C. for 60 s using the primers; Forward nested5′-AGGGGGGTGAAGTCGTAACAAG-3′ (SEQ ID NO: 28) and Reversed nested5′-GTCTGATAAACCTGAGGTCGGA-3′ (SEQ ID NO: 29). Of the final reactionvolume, 5 μL was separated on a 1% agarose gel containing ethidiumbromide and bands were visualized in UV-light.

In each PCR analysis, DNA purified from an in vitro grown culture of B.afzelii strain K78 was used as a positive control template. In addition,PBS was used instead of extracted DNA as negative control. Fivemicroliters of the final product was separated on a 1% agarose gelcontaining ethidium bromide and bands were visualized in UV-light.

ELISA with the Invariable Region 6 (IR6) of the Variable MajorProtein-Like Sequence E Protein (VlsE)

A biotinylated 25-mer peptide (MKKDDQIAAAMVLRGMAKDGQFALK) (SEQ ID NO:30) derived from the sequence of B. garinii strain IP90 was used foranalysis (Liang F T, et al. (1999) J Immunol. 163:5566-73). Streptavidinpre-coated 96-well ELISA plates (Nunc, Denmark) were coated with 100μL/well (1 μg/mL) biotinylated peptide in PBS supplemented with 0.1%Tween 20 (PBS/0.1T). The plates were incubated overnight at 4° C. Aftercoating with the peptide, the plates were washed once with PBS/0.1T. Theplates were then blocked for one hour at room temperature (RT) with 100μL/well of PBS+2% BSA, before being washed again with PBS/0.1T.Reactivity of post-challenge sera to the peptide was tested at 1:200,1:400 and 1:800 dilutions in PBS+1% BSA. Plates were incubated for 90min at RT before being washed three times with PBS/0.1T. Each well thenreceived 50 μL of 1.3 μg/mL polyclonal rabbit anti-mouse IgG conjugatedto HRP (Dako, Denmark) in PBS+1% BSA. The plates were then incubated for1 h at RT. After three washes with PBS/0.1T, ABTS (50 μl/well) was addedas substrate (Sigma-Aldrich, USA) and color was allowed to develop for30 min. Absorbance was measured at 405 nm. All sera were tested induplicate; negative controls included PBS instead of sera, as well asplates not coated with the peptide. Sera from mice shown to be culturepositive for B. afzelii infection were used as positive controls.

Results

Levels of Protection in the Tick Challenge Method

Extremely significant levels of protection (p-value 0.001) were seen forall five stabilized OspA B. afzelii fragments at both of the dosestested (30 μg and 5 μg, see Table 2). The high infection rates in thePBS control group indicate that the ticks were infected with highfrequency. Additionally, the positive control, non-lipidated full-lengthOspA from B. afzelii strain K78, was very protective. Together thesecontrol groups indicate the high reliability of the experimentalreadout.

Protection data from the seven experiments are combined and summarizedin Table 2. The two methods employed to verify infection, namely ELISAand PCR, gave virtually identical results (not shown), demonstrating therobustness of these methods for assessment of infection in the tickchallenge method.

Example 3. Protection of Mice from Infection with B. afzelii by the TickChallenge Method by Immunization with Lipidated Mutant Serotype 2 OspAFragments

Experimental Procedures

Cloning and Expression of Lipidated his-Tagged Mutant OspA FragmentProteins

The serotype 2 mutant OspA fragments with cysteine bond types 2, 3 and 4(SEQ ID NOs: 142, 143 and 144, respectively) were modified by theaddition of a lipidation signal sequence derived from OspA (SEQ ID NO:14) and followed directly C-terminally by a CKQN peptide (SEQ ID NO:211) to provide an N-terminal cysteine for lipidation. All mutant OspAfragments were C-terminally histidine-tagged for purification purposes.Gene fragments were cloned into the pET28b(+) vector (Novagen), a vectorcontaining a Kanamycin resistance cassette as well as a T7 promoter. Thelipidated monomers were expressed in BL21 Star™(DE3) cells (Invitrogen)and after induction by IPTG, the growth temperature of the cells waslowered from 37° C. to 25° C. to promote efficient post-translationalprocessing of the proteins. Cells were collected after 4 h bycentrifugation and the pellet was stored at −70° C. for up to 12 monthsprior to further processing.

Purification of Lipidated his-Tagged Wild-Type and Mutant OspA FragmentMonomer Proteins

Cells were disrupted mechanically by high-pressure homogenization andthe lipidated His-tagged OspA fragment monomer polypeptides wereenriched in the lipid phase by phase separation, using Triton X-114 asdetergent. Subsequently, the diluted detergent phase (20 to 30 fold) wasapplied to a Ni-sepharose column (Ni Sepharose™ 6 Fast Flow; GEHealthcare) and the lipidated His-tagged OspA fragments were eluted byImidazole gradient (0-250 mM) elution. Pooled fractions were furtherpurified over a gel filtration column (Superdex 200, GE Healthcare)followed by a buffer exchange column (Sephadex G-25, GE Healthcare).Lipidated His-tagged OspA fragment peaks were pooled on the basis of theanalytical size exclusion column and reversed phase chromatography.After sterile filtration, the purified proteins were stored at −20° C.until formulation.

Immunization of Mice

Three lipidated mutant OspA proteins (Lip-S2D2-His, Lip-S2D3-His andLip-S2D4-His) were expressed and purified as described above. In vivoprotection studies were performed also as above using PBS andnon-lipidated full-length serotype 2 OspA as negative and positivecontrols, respectively. All immunogens were formulated with 0.15%Al(OH)₃. Mice were injected subcutaneously three times at two weekintervals with formulations containing 3.0 μg, 1.0 μg or 0.3 μg antigenand challenged with infected ticks two weeks after the lastimmunization. Mice were sacrificed six weeks following tick challengeand infection was assessed.

Results

Levels of Protection in the Tick Challenge Method

All three lipidated mutant OspA fragments conferred extremelysignificant levels of protection (p-value≦0.001) from B. afzeliichallenge even at the lowest tested dose (Table 3). Infection rates inthe PBS groups were high, indicating that the ticks were infected to ahigh frequency. The positive control antigen, full-length non-lipidatedOspA from B. afzelii strain K78, was also very protective. Together,these control groups indicate the high reliability of the method ofinfection and thus give high credibility to the results observedfollowing immunization with the lipidated mutant OspA fragments.

Example 4. Protection of Mice from Borrelia Infection by Immunizationwith Lipidated his-Tagged Mutant OspA Fragment Heterodimers of DifferentSerotypes

Experimental Procedures

Cloning and Expression of Lipidated his-Tagged Mutant OspA FragmentHeterodimers

The mutant OspA fragment monomers from B. burgdorferi s.s. strain B31,B. afzelii strain K78, B. garinii strain PBr, B. bavariensis strain PBi,B. garinii strain PHEi and B. garinii strain DK29 were codon-optimizedfor E. coli expression by GenScript, USA. The hLFA-1-like epitope (aa164-174, SEQ ID NO: 17) of the OspA from B. burgdorferi s.s. strain B31was replaced by a non-hLFA-1-like sequence NFTLEGKVAND from B. afzeliistrain K78 (SEQ ID NO: 18). The lipidation signal sequence added to themutant OspA fragment heterodimers was derived from the E. coli majorouter membrane lipoprotein, Lpp, and was followed directly C-terminallyby a CSS peptide (SEQ ID NO: 210) to provide an N-terminal cysteine forlipidation. The mutant OspA fragment heterodimers were generated byfusing different mutant OspA fragment monomers as described above via a21 amino acid linker sequence, originating from two separate loopregions of the N-terminal half of OspA from B. burgdorferi s.s. strainB31 (“LN1”; aa 65-74 and aa 42-53 with an amino acid exchange of D53S,SEQ ID NO: 184). The heterodimers were constructed with a His-tag forpurification purposes. Gene fragments were cloned into the pET28b(+)vector (Novagen), a vector containing a Kanamycin resistance cassette aswell as a T7 promoter. The lipoproteins of the stabilized heterodimerswere expressed in BL21 Star™(DE3) cells (Invitrogen) and after inductionby IPTG, the growth temperature of the cells was lowered from 37° C. to25° C. to promote efficient post-translational processing of theproteins. Cells were collected after 4 h by centrifugation and thepellet was stored at −70° C. for up to 12 months prior to furtherprocessing.

Purification of Lipidated his-Tagged Mutant OspA Fragment Heterodimers

Cells were disrupted mechanically by high-pressure homogenization andthe lipidated His-tagged mutant OspA fragment heterodimers were enrichedin the lipid phase by phase separation, using Triton X-114 as detergent.Subsequently, the diluted detergent phase (20 to 30 fold) was applied toa Ni-sepharose column (Ni Sepharose™ 6 Fast Flow; GE Healthcare) and thelipidated His-tagged OspA heterodimers were eluted by Imidazole gradient(0-250 mM) elution. Pooled fractions were further purified over a gelfiltration column (Superdex 200, GE Healthcare) followed by a bufferexchange column (Sephadex G-25, GE Healthcare). The lipidated His-taggedmutant OspA heterodimer peaks were pooled on the basis of the analyticalsize exclusion column and reversed phase chromatography. After sterilefiltration, the purified heterodimers were stored at −20° C. untilformulation.

Immunization of Mice

Female C3H/HeN mice (Janvier, France) were used for all studies. Priorto each challenge, groups of ten 8-week-old mice were bled via thefacial vein and pre-immune sera were prepared and pooled. Threesubcutaneous (s.c.) immunizations of 100 μL each were administered attwo week intervals. Each dose contained either 2 μg or 5 μg of theheterodimer protein Lip-S1D1-S2D1-His (SEQ ID NO: 49), or 1.0 μg or 2.5μg of the respective monomer proteins, formulated with aluminiumhydroxide (Al(OH)₃) at a final concentration of 0.15%. One week afterthe third immunization, blood was collected from the facial vein andhyper-immune sera were prepared. In each experiment, one group immunizedwith Al(OH)₃ alone was included as a negative control. All animalexperiments were conducted in accordance with Austrian law (BGB1 Nr.501/1989) and approved by “Magistratsabteilung 58”.

Tick Challenge of Immunized Mice and Collection of Sera and Tissues(Herein Referred to Also as “Tick Challenge Method”)

In order to challenge the immunized mice with B. afzelii, the hair ofthe back of each mouse was removed with Veet® Cream (Reckitt Benckiser)and a small ventilated container was glued to the skin with super glue(Pattex). Thereafter, one or two I. ricinus nymphs infected with B.afzelii, strain IS1, were applied per mouse, allowed to attach and feeduntil they were fully engorged and dropped off. The feeding status wasmonitored for each individual tick and only mice from which at least onefully fed tick was collected were included in the final readout.

Needle Challenge of Immunized Mice with In Vitro Grown Borrelia

Two weeks after the last immunization, the mice were challenged s.c.with Borrelia diluted in 100 μL Borrelia growth medium (BSK II). Thechallenge doses were strain-dependent, the virulence of the individualstrains being assessed by challenge experiments for determination ofID₅₀. Doses employed for needle challenge experiments ranged from 20 to50 times the ID₅₀.

Sacrifice of Mice and Collection of Material

Four weeks after needle challenge with B. burgdorferi s.s. or six weeksafter tick challenge with B. afzelii, mice were sacrificed by cervicaldislocation. The blood was collected by orbital bleeding and final serawere prepared and used for VlsE ELISA to determine infection status. Inaddition, one ear from each mouse was collected, and DNA was extractedand subjected to qPCR for identification of Borrelia. The finalinfection readout was based on two different analyses (qPCR targetingrecA and VlsE ELISA).

ELISA with the Invariable Region 6 (IR6) of VlsE

A biotinylated 25-mer peptide (MKKDDQIAAAMVLRGMAKDGQFALK) (SEQ ID NO:30) derived from the sequence of B. garinii strain IP90 was used for theanalysis (Liang F T, Alvarez A L, Gu Y, Nowling J M, Ramamoorthy R,Philipp M T. An immunodominant conserved region within the variabledomain of VlsE, the variable surface antigen of Borrelia burgdorferi. JImmunol. 1999; 163:5566-73). Streptavidin pre-coated 96-well ELISAplates (Nunc), were coated with 100 μL/well (1 μg/mL) peptide in PBSsupplemented with 0.1% Tween (PBS/0.1T). The plates were incubatedovernight at 4° C. After coating with the peptide, the plates werewashed once with PBS/0.1T. The plates were then blocked for one hour atroom temperature (RT) with 100 μL/well of PBS+2% BSA, before beingwashed again with PBS/0.1T. Reactivity of post-challenge sera to thepeptide was tested at 1:200, 1:400 and 1:800 dilutions in PBS+1% BSA.Plates were incubated for 90 min at RT before being washed three timeswith PBS/0.1T. Each well then received 50 μL of 1.3 μg/mL polyclonalrabbit anti-mouse IgG conjugated to HRP (Dako) in PBS+1% BSA. The plateswere then incubated for 1 h at RT. After three washes with PBS/0.1T,ABTS (50 μL/well) was added as substrate (Sigma-Aldrich) and color wasallowed to develop for 30 min Absorbance was measured at 405 nm. Allsera were tested in duplicate. Negative controls included PBS instead ofsera as well as plates not coated with the peptide. Sera from mice shownto be culture positive for B. afzelii infection were used as positivecontrols.

qPCR Targeting recA

Oligonucleotide primers were designed for the recA gene in a manner thatthey could be used in qPCR for identification of all relevant Borreliaspecies causing Lyme borreliosis (forward: CATGCTCTTGATCCTGTTTA, SEQ IDNO: 213 reverse: CCCATTTCTCCATCTATCTC, SEQ ID NO: 215). The recAfragment was cloned from the B. burgdorferi s.s. strain N40 intopET28b(+), to be used as standard in each reaction. The chromosomal DNAextracted from mouse ears was diluted 1:8 in water in order to reducematrix effects observed with undiluted DNA. A master mix consisting of10 μL, SSoAdvanced™ SYBR® Green Supermix, 0.3 μL, of each primer (10μM), and 7.4 μL, water was prepared for each experiment. Eighteen μL, ofmaster mix was mixed with 2 μL, of the diluted DNA extracted from eitherbladder or ear in micro-titer plates and the DNA was amplified using aCFX96 real-time PCR detection system (Bio-Rad, USA). The DNA wasdenatured for 3 minutes at 95° C., followed by 50 cycles of 15 secondsat 95° C. and 30 seconds at 55° C. After amplification, the DNA wasprepared for the melting curve analysis by denaturation for 30 secondsat 95° C. followed by 2 minutes at 55° C. The melting curve analysis wasperformed by 5 seconds incubation at 55° C., with a 0.5° C. increase percycle, and 5 seconds at 95° C. On each plate, four no-template controls(NTC) were included as well as a standard curve in duplicate withtemplate copy numbers ranging from 10 to 10,000.

Results

The lipidated His-tagged mutant OspA fragment heterodimer(Lip-S1D1-S2D1-His) was tested for protective capacity in six separateexperiments. Mice were challenged with either the B. burgdorferi s.s.strain N40 (needle challenge) or the B. afzelii strain IS1 (tickchallenge) in three experiments each. All experiments included miceimmunized with the individual respective lipidated His-tagged mutantOspA monomers as positive control groups (Lip-S1D1-His and Lip-S2D1-His)and mice immunized with adjuvant alone as a negative control group. Forchallenge with ticks, 1-2 ticks were applied per mouse and only micefrom which at least one tick fed until fully engorged were included inthe final readout. However, no distinction was made between mice fromwhich one or two fully fed ticks were collected. The protection datafrom the six experiments are summarized in Table 4.

The lipidated His-tagged OspA heterodimer (Lip-S1D1-S2D1-His) showedhighly statistically-significant protection (Fisher's exact test,two-tailed) in all six experiments against both challenge species ascompared to the negative control group. The infectious status of eachmouse was determined using either VlsE ELISA alone (experiments 1-3) orin combination with recA qPCR (experiments 4-6). In cases where bothmethods were used, a mouse was regarded as infected when at least onemethod gave a positive result. The level of protection conferred by thelipidated His-tagged mutant OspA fragment heterodimer(Lip-S1D1-S2D1-His) was equal to or better than the protection conferredby either of the lipidated His-tagged mutant OspA fragment monomersindividually.

Example 5. Protection of Mice from Infection with Borrelia byImmunization with Lipidated Non-his-Tagged Mutant OspA FragmentHeterodimers of Different Serotypes

Cloning and Expression of Lipidated Non-his-Tagged Mutant OspA FragmentHeterodimers

The constructs made as described in Example 4 were used for thegeneration of His-less constructs by the introduction of a stop codon byPCR amplification. Gene fragments were cloned into the pET28b(+) vector(Novagen), a vector containing a Kanamycin resistance cassette as wellas a T7 promoter. The lipoproteins of the stabilized heterodimers wereexpressed in BL21 Star™(DE3) cells (Invitrogen) and after induction byIPTG, the growth temperature of the cells was lowered from 37° C. to 25°C. to promote efficient post-translational processing of the proteins.Cells were collected after 4 h by centrifugation and the pellet wasstored at −70° C. for up to 12 months prior to further processing.

Purification of Lipidated Non-his-Tagged Mutant OspA FragmentHeterodimers

Cells were disrupted mechanically by high-pressure homogenization andthe lipidated mutant OspA fragment heterodimers were enriched in thelipid phase by phase separation, using Triton X-114 as detergent.Subsequently, the diluted detergent phase was subjected to anionexchange chromatography. The resulting flow-through was subjected tocation exchange chromatography and the lipidated proteins eluted fromthe column. The eluate was subjected to further purification over a gelfiltration column (Superdex 200, GE Healthcare) followed by a bufferexchange column (Sephadex G-25, GE Healthcare). The lipidated mutantOspA heterodimer peaks were pooled on the basis of the analytical sizeexclusion column and reversed phase chromatography. After sterilefiltration, the purified heterodimers were stored at −20° C. untilformulation.

Immunization of Mice

Female C3H/HeN mice will be used for all studies. Prior to eachchallenge, groups of ten 8-week-old mice will be bled via the facialvein and pre-immune sera will be prepared and pooled. Three s.c.immunizations of 100 μL each will be administered at two week intervals.Each dose will contain 5 μg of the respective heterodimer proteins:Lip-S1D1-S2D1 (SEQ ID NO: 186), Lip-S4D1-S3D1 (SEQ ID NO: 194) andLip-S5D1-S6D1 (SEQ ID NO: 190) or 2.5 μg of the respective monomerproteins, formulated with aluminium hydroxide (Al(OH)₃) at a finalconcentration of 0.15%. One week after the third immunization, bloodwill be collected from the facial vein and hyper-immune sera will beprepared. In each experiment, one group immunized with PBS formulatedwith Al(OH)₃ will be included as a negative control. All animalexperiments will be conducted in accordance with Austrian law (BGB1 Nr.501/1989) and approved by “Magistratsabteilung 58”.

Needle Challenge of Immunized Mice with In Vitro Grown Borrelia

Two weeks after the last immunization, the mice will be challenged s.c.with Borrelia diluted in 100 μL Borrelia growth medium (BSKII). Thechallenge doses are strain-dependent, the virulence of the individualstrains will require assessment by challenge experiments fordetermination of ID₅₀. Doses employed for needle challenge experimentswill range from 20 to 50 times the ID₅₀. Four weeks after needlechallenge, mice will be sacrificed and blood and tissues will becollected for readout methods to determine the infection status.

SEQUENCES

SEQ ID NO: 1  S2D0-His amino acids of positions 131-273 of Borreliaafzelii strain K78, OspA serotype 2, wild-typesequence, C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214)ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKG LEHHHHHH SEQ ID NO: 2 S2D1-His  aa 131-273 of Borrelia afzelii strain K78, OspAserotype 2 with disulfide bond type 1 (aa 182and 269), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTL EVK CGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDEL C NALKG  LEHHHHHHSEQ ID NO: 3  S2D2-His aa 131-273 of Borrelia afzelii strain K78, OspAserotype 2 with disulfide bond type 2 (aa 182and 272), C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTL EVK CGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNA C KG  LEHHHHHHSEQ ID NO: 4  S2D3-His aa 131-273 of Borrelia afzelii strain K78, OspA  serotype 2 with disulfide bond type 3 (aa 244and 259), C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTIS VNSKKTTQLVFTKQDTI CVQKYDSAGTNLEGT C VEIKTLDELKNALKG LEHHHHHH  SEQ ID NO: 5  S2D4-His aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 4 (aa 141 and 241), C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214)  ELSAKTMTRE CGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTIS VNSKKTTQLVFTKQ CTITVQKYDSAGTNLEGTAVEIKTLDELKNALKG LEHHHHHH SEQ ID NO: 6  S2D5-His aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 5 (aa 165 and 265), C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKN C TLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKT C DELKNALKG LEHHHHHH SEQ ID NO: 7  S2D6-His  aa 131-273 of Borrelia afzelii strain K78, OspAsero type 2 with disulfide bond type 6 (aa 185 and 272), C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTL EVKEGT CTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNA C KG LEHHHHHH SEQ ID NO: 8  S2D7-His  aa 131-273 of Borrelia afzelii strain K78, OspAserotype 2 with disulfide bond type 7 (aa 199 and 223), C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTL EVKEGTVTLSKEIAKSGEVT CALNDTNTTQATKKTGAWDSKTST C TISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKG LEHHHHHH  SEQ ID NO: 9 S2D8-His  aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 8 (aa 243 and 262), C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTIS VNSKKTTQLVFTKQDT CTVQKYDSAGTNLEGTAVE C KTLDELKNALKG LEHHHHHH  SEQ ID NO: 10  S2D9-His aa 131-273 of Borrelia afzelii strain K78, OspAserotype 2 with disulfide bond type 9 (aa 184 and 204), C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTL EVKEG CVTLSKEIAKSGEVTVALND C NTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKG  LEHHHHHHSEQ ID NO: 11  S2D10-His aa 131-273 of Borrelia afzelii strain K78, OspAserotype 2 with disulfide bond type 10 (aa 201 and 214), C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTL EVKEGTVTLSKEIAKSGEVTVAC NDTNTTQATKKT C AWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKG LEHHHHHH SEQ ID NO: 12  S2D11-His aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 11 (aa 246 and 259), C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTIS VNSKKTTQLVFTKQDTITV CKYDSAGTNLEGT C VEIKTLDELKNALKG LEHHHHHH  SEQ ID NO: 13  S2D12-His aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 12 (aa 167 and 178), C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFT C EGKVANDKVT CEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKG LEHHHHHH SEQ ID NO: 14  Borrelia OspA lipidation signal  MKKYLLGIGLILALIA SEQ ID NO: 15  Borrelia OspB lipidation signal  MRLLIGFALALALIG SEQ ID NO: 16  E. coli lpp lipidation signal  MKATKLVLGAVILGSTLLAG SEQ ID NO: 17  hLFA-1-like sequence from B. burgdorferi s.s. strain B31  GYVLEGTLTAE  SEQ ID NO: 18 Non-hLFA-1-like sequence from B. afzelii  strain K78  NFTLEGKVAND SEQ ID NO: 19  B. afzelii (strain K78; OspA serotype 2) OspA MKKYLLGIGLILALIACKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIELKGTSDKDNGSGVLEGTKDDKSKAKLTIADDLSKTTFELFKEDGKTLVSRKVSSKDKTSTDEMFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 20 B. burgdorferi s.s. (strain B31, OspA serotype  1) OspA MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALK SEQ ID NO: 21 B. garinii (strain PBr, OspA serotype 3) OspA MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKSNGSGVLEGEKADKSKAKLTISQDLNQTTFEIFKEDGKTLVSRKVNSKDKSSTEEKFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELKAALK SEQ ID NO: 22 B. bavariensis (strain PBi, OspA serotype 4)  OspA MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLMATVDKLELKGTSDKSNGSGTLEGEKSDKSKAKLTISEDLSKTTFEIFKEDGKTLVSKKVNSKDKSSIEEKFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELKNALK SEQ ID NO: 23 B. garinii (strain PHei, OspA serotype 5) OspA MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKNNGSGTLEGEKTDKSKVKLTIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTEEKFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 24 B. garinii (strain DK29, OspA serotype 6) OspA MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMTVLVSKEKDKDGKYSLEATVDKLELKGTSDKNNGSGTLEGEKTDKSKVKSTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 25 B. garinii (strain T25, OspA serotype 7) OspA MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLEATVDKLELKGTSDKNNGSGVLEGVKAAKSKAKLTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKFNDKGKLSEKVVTRANGTRLEYTEIQNDGSGKAKEVLKSLTLEGTLTADGETKLTVEAGTVTLSKNISESGEITVELKDTETTPADKKSGTWDSKTSTLTISKNSQKTKQLVFTKENTITVQKYNTAGTKLEGSPAEIKDLEALKAALK SEQ ID NO: 26 Borrelia nested pcr detection forward primer 1  GTATGTTTAGTGAGGGGGGTG SEQ ID NO: 27  Borrelia nested pcr detection reverse primer 1 GGATCATAGCTCAGGTGGTTAG  SEQ ID NO: 28 Borrelia nested pcr detection forward primer 2  AGGGGGGTGAAGTCGTAACAAG SEQ ID NO: 29  Borrelia nested pcr detection reverse primer 2 GTCTGATAAACCTGAGGTCGGA  SEQ ID NO: 30  25-mer peptide MKKDDQIAAAMVLRGMAKDGQFALK  SEQ ID NO: 31  Mouse cathelin RLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE  SEQ ID NO: 32  5′-(dIdC)₁₃-3′dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC  dIdC dIdC dIdC dIdC SEQ ID NO: 33  KLK peptide  KLKLLLLLKLK  SEQ ID NO: 34 B. afzelii (strain K78, serotype 2), OspA aa  126-273 FNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELK NALK SEQ ID NO: 35 B. afzelii (strain K78, serotype 2), OspA aa  131-273 ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 36 peptide linker  GGGGGGGG  SEQ ID NO: 37  peptide linker 2  GGGGGGGGGGGG SEQ ID NO: 38  peptide linker 3  GAGA  SEQ ID NO: 39  peptide linker 4 GAGAGAGA  SEQ ID NO: 40  peptide linker 5  GAGAGAGAGAGA  SEQ ID NO: 41 peptide linker 6  GGGSGGGS  SEQ ID NO: 42  peptide linker 7 GGGSGGGSGGGS  SEQ ID NO: 43  S1D4-S2D4_aa Heterodimer fusion protein of OspA serotypes 1 and 2 both with disulfide bond type 4, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) FNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 44  Lip-S1D4-S2D4_nt Coding sequence for fusion proteins of OspA serotypes 1 and 2 both with disulfide bond type4, E. coli lpp lipidation signal, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAA AACGCCCTGAAASEQ ID NO: 45  Lip-S1D4-S2D4_His_aa Heterodimer fusion protein of OspA serotypes 1 and 2 both with disulfide bond type 4, N- terminal CSS for addition of lipids, N-terminal lipidation, LN1 peptide linker (SEQ ID NO: 184),aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18),C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 46 Lip-S1D4-S2D4_His_nt  Coding sequence for heterodimer fusion protein of OspA serotypes 1 and 2 both with disulfide bond type 4, E. coli lpp lipidation signal, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), C-terminalHis tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCCTCGAGCACCACCACCACCACCAC SEQ ID NO: 47  S1D1-S2D1_aa Heterodimer fusion protein of OspA serotype 1 and OspA serotype 2 with disulfide bond type 1,LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) FNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK SEQ ID NO: 48  Lip-S1D1-S2D1_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 1 and OspA serotype 2 with disulfide bond type 1, E. coli lpp lipidation signal, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGT AACGCCCTGAAASEQ ID NO: 49  Lip-S1D1-S2D1_His_aa Heterodimer fusion protein of OspA serotype 1 and OspA serotype 2 with disulfide bond type 1,N-terminal CSS for addition of lipids, N- terminal lipidation, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGLEHHHHHH SEQ ID NO: 50 Lip-S1D1-S2D1_His_nt  Coding sequence for heterodimer fusion protein of OspA serotype 1 and OspA serotype 2 with disulfide bond type 1, E. coli lpp lipidation signal, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCCTCGAGCACCACCACCACCACCAC SEQ ID NO: 51  S3D4-S4D4_aa Heterodimer fusion protein of OspA serotype 3 and OspA serotype 4 with disulfide bond type 4, LN1 peptide linker (SEQ ID NO: 184) FNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALK SEQ ID NO: 52  Lip-S3D4-S4D4_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 3 and OspA serotype 4 with disulfide bond type 4, E. coli lpp lipidation signal, LN1 peptide  linker (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTG AAAAATGCTCTGAAGSEQ ID NO: 53  Lip-S3D4-S4D4_His_aa Heterodimer fusion protein of OspA serotype 3 and OspA serotype 4 with disulfide bond type 4, N-terminal CSS for addition of lipids, N-terminal lipidation, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCSSFNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 54 Lip-S3D4-S4D4_His_nt  Coding sequence for heterodimer fusion protein of OspA serotype 3 and OspA serotype 4 with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAATGCTCTGAAGGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 55  S3D1-S4D1_aa Heterodimer fusion protein of OspA serotypes 3 and 4 both with disulfide bond type 1, LN1 peptide linker (SEQ ID NO: 184) FNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALK SEQ ID NO: 56  Lip-S3D1-S4D1_nt Coding sequence for intermediate and finalheterodimer fusion proteins of OspA serotypes 3and 4 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker  (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTG TGTAATGCTCTGAAGSEQ ID NO: 57  Lip-S3D1-S4D1_His_aa Heterodimer fusion protein of OspA serotypes 3 and 4 both with disulfide bond type 1, E. colilpp lipidation signal, N-terminal CSS for addition of lipids, N-terminal lipidation, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGLEHHHHHH SEQ ID NO: 58 Lip-S3D1-S4D1_His_nt  Coding sequence for heterodimer fusion protein of OspA serotypes 3 and 4 both with disulfidebond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAATGCTCTGAAGGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 59  S5D4-S6D4_aa Heterodimer fusion protein OspA serotypes 5 and 6 both with disulfide bond type 4, LN1 peptide  linker (SEQ ID NO: 184) FNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 60  Lip-S5D4-S6D4_nt Coding sequence for intermediate and final heterodimer fusion proteins OspA serotypes 5 and 6 both with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker  (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTG AAGAATGCTCTGAAASEQ ID NO: 61  Lip-S5D4-S6D4_His_aa Heterodimer fusion protein OspA serotypes 5 and 6 both with disulfide bond type 4, N-terminal CSS for addition of lipids, N-terminal lipidation, LN1 peptide linker (SEQ ID NO: 184),C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGLEHHHHHH SEQ ID NO: 62 Lip-S5D4-S6D4_His_nt  Coding sequence for heterodimer fusion protein OspA serotypes 5 and 6 both with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptidelinker (SEQ ID NO: 184), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 63  S5D1-S6D1_aa Heterodimer fusion protein of OspA serotypes 6 both with disulfide bond type 1, LN1 peptide  linker (SEQ ID NO: 184) FNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 64  Lip-S5D1-S6D1_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotypes 6 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTG TGTAATGCTCTGAAASEQ ID NO: 65  Lip-S5D1-S6D1_His_aa Heterodimer fusion protein of OspA serotypes 6 both with disulfide bond type 1, N-terminal CSS for addition of lipids, N-terminal lipidation, LN1 peptide linker (SEQ ID NO: 184), C-terminalHis tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGLEHHHHHH SEQ ID NO: 66 Lip-S5D1-S6D1_His_nt  Coding sequence for heterodimer fusion protein of OspA serotypes 6 both with disulfide bondtype 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptidelinker (SEQ ID NO: 184), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 67  S2D4-S1D4_aa Heterodimer fusion protein of OspA serotypes 2 and 1 both with disulfide bond type 4, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-likesequence NFTLEGKVAND (SEQ ID NO: 18) FNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALK SEQ ID NO: 68  Lip-S2D4-S1D4_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotypes 2 and 1 both with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence  NFTLEGKVAND (SEQ ID NO: 18) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAA AATGCACTGAAASEQ ID NO: 69  Lip-S2D4-S1D4_His_aa Heterodimer fusion protein of OspA serotypes 2 and 1 both with disulfide bond type 4, N- terminal CSS for addition of lipids, N-terminallipidation, LN1 peptide linker (SEQ ID NO: 184),aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCSSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGLEHHHHHH SEQ ID NO: 70 Lip-S2D4-S1D4_His_nt  Coding sequence for heterodimer fusion protein of OspA serotypes 2 and 1 both with disulfidebond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-likesequence NFTLEGKVAND (SEQ ID NO: 18), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 71  S2D1-S1D1_aa Heterodimer fusion protein of OspA serotypes 2 and 1 both with disulfide bond type 1, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) FNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALK SEQ ID NO: 72  Lip-S2D1-S1D1_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotypes 2 and 1 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker(SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND  (SEQ ID NO: 18) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGT AATGCACTGAAASEQ ID NO: 73  Lip-S2D1-S1D1_His_aa Heterodimer fusion protein of OspA serotypes 2 and 1 both with disulfide bond type 1, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), N-terminal lipidation, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCSSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGLEHHHHHH SEQ ID NO: 74 Lip-S2D1-S1D1_His_nt  Coding sequence for heterodimer fusion protein of OspA serotypes 2 and 1 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-likesequence NFTLEGKVAND (SEQ ID NO: 18), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 75  S4D4-S3D4_aa Heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfide bond type 4, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184) FNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALK SEQ ID NO: 76  Lip-S4D4-S3D4_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotypes 4 and 3 both with disulfide bond type 4, E. coli 1pp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTG AAGGCGGCACTGAAASEQ ID NO: 77  Lip-S4D4-S3D4_His_aa Heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfide bond type 4, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCSSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGLEHHHHHH SEQ ID NO: 78 Lip-S4D4-S3D4_His_nt  Coding sequence for heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfidebond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 79  S4D1-S3D1_aa Heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfide bond type 1, LN1 peptide linker (SEQ ID NO: 184) FNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK SEQ ID NO: 80  Lip-S4D1-S3D1_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotypes 4 and 3 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker  (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTG TGTGCGGCACTGAAASEQ ID NO: 81  Lip-S4D1-S3D1_His_aa Heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfide bond type 1, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGLEHHHHHH SEQ ID NO: 82 Lip-S4D1-S3D1_His_nt  Coding sequence for heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 83  S6D4-S5D4_aa Heterodimer fusion protein of OspA serotypes 6 and 5 both with disulfide bond type 4, LN1 peptide linker (SEQ ID NO: 184) FNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 84  Lip-S6D4-S5D4_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotypes 6 and 5 both with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker  (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTG AAGAATGCTCTGAAASEQ ID NO: 85  Lip-S6D4-S5D4_His_aa Heterodimer fusion protein of OspA serotypes 6 and 5 both with disulfide bond type 4, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCSSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGLEHHHHHH SEQ ID NO: 86 Lip-S6D4-S5D4_His_nt  Coding sequence for heterodimer fusion protein of OspA serotypes 6 and 5 both with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 87  S6D1-S5D1_aa Heterodimer fusion protein of OspA serotypes 6 and 5 both with disulfide bond type 1, LN1 peptide linker (SEQ ID NO: 184) FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 88  Lip-S6D1-S5D1_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotypes 6 and 5 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker  (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTG TGTAATGCTCTGAAASEQ ID NO: 89  Lip-S6D1-S5D1_His_aa Heterodimer fusion protein of OspA serotypes 6 and 5 both with disulfide bond type 1, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCSSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGLEHHHHHH SEQ ID NO: 90 Lip-S6D1-S5D1_His_nt  Coding sequence for heterodimer fusion protein of OspA serotypes 6 and 5 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 91  S1D4-S2D1_aa Heterodimer fusion protein of OspA serotype 1 with disulfide bond type 4 and OspA serotype 2 with disulfide bond type 1, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) FNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK SEQ ID NO: 92  Lip-S1D4-S2D1_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 1 with disulfide bond type 4 and OspA serotype 2 with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184),aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGT AACGCCCTGAAASEQ ID NO: 93  Lip-S1D4-S2D1_His_aa Heterodimer fusion protein of OspA serotype 1 with disulfide bond type 4 and OspA serotype 2 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND(SEQ ID NO: 18), N-terminal lipidation, C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGLEHHHHHH SEQ ID NO: 94 Lip-S1D4-S2D1_His_nt  Coding sequence for heterodimer fusion protein of OspA serotype 1 with disulfide bond type 4 and OspA serotype 2 with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCCTCGAGCACCACCACCACCACCAC SEQ ID NO: 95  S1D1-S2D4_aa Heterodimer fusion protein of OspA serotype 1 with disulfide bond type 1 and OspA serotype 2 with disulfide bond type 4, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) FNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 96  Lip-S1D1-S2D4_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 1 with disulfide bond type 1 and OspA serotype 2 with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184),aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAA AACGCCCTGAAASEQ ID NO: 97  Lip-S1D1-S2D4_His_aa Heterodimer fusion protein of OspA serotype 1 with disulfide bond type 1 and OspA serotype 2 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND(SEQ ID NO: 18), N-terminal lipidation, C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 98 Lip-S1D1-S2D4_His_nt  Coding sequence for heterodimer fusion protein of OspA serotype 1 with disulfide bond type 1 and OspA serotype 2 with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCCTCGAGCACCACCACCACCACCAC SEQ ID NO: 99  S3D4-S4D1_aa Heterodimer fusion protein of OspA serotype 3 with disulfide bond type 4 and OspA serotype 4 with disulfide bond type 1, LN1 peptide linker  (SEQ ID NO: 184) FNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALK SEQ ID NO: 100  Lip-S3D4-S4D1_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 3 with disulfide bond type 4 and OspA serotype 4 with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTG TGTAATGCTCTGAAGSEQ ID NO: 101  Lip-S3D4-S4D1_His_aa Heterodimer fusion protein of OspA serotype 3 with disulfide bond type 4 and OspA serotype 4 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation, C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGLEHHHHHH SEQ ID NO: 102 Lip-S3D4-S4D1_His_nt  Coding sequence for heterodimer fusion protein of OspA serotype 3 with disulfide bond type 4 and OspA serotype 4 with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag  (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAATGCTCTGAAGGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 103  S3D1-S4D4_aa Heterodimer fusion protein of OspA serotype 3 with disulfide bond type 1 and OspA serotype 4 with disulfide bond type 1, LN1 peptide linker  (SEQ ID NO: 184) FNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALK SEQ ID NO: 104  Lip-S3D1-S4D4_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 3 with disulfide bond type 1 and OspA serotype 4 with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTG AAAAATGCTCTGAAGSEQ ID NO: 105  Lip-S3D1-S4D4_His_aa Heterodimer fusion protein of OspA serotype 3 with disulfide bond type 1 and OspA serotype 4 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation, C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 106 Lip-S3D1-S4D4_His_nt  Coding sequence for heterodimer fusion protein of OspA serotype 3 with disulfide bond type 1 and OspA serotype 4 with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag  (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAATGCTCTGAAGGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 107  S5D4-S6D1_aa Heterodimer fusion protein of OspA serotype 5 with disulfide bond type 4 and OspA serotype 6 with disulfide bond type 1, LN1 peptide linker  (SEQ ID NO: 184) FNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 108  Lip-S5D4-S6D1_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 5 with disulfide bond type 4 and OspA serotype 6 with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTG TGTAATGCTCTGAAASEQ ID NO: 109  Lip-S5D4-S6D1_His_aa Heterodimer fusion protein of OspA serotype 5 with disulfide bond type 4 and OspA serotype 6 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation, C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGLEHHHHHH SEQ ID NO: 110 Lip-S5D4-S6D1_His_nt  Coding sequence for heterodimer fusion protein of OspA serotype 5 with disulfide bond type 4 and OspA serotype 6 with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag  (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 111  S5D1-S6D4_aa Heterodimer fusion protein of OspA serotype 5 with disulfide bond type 1 and OspA serotype 6 with disulfide bond type 4, LN1 peptide linker  (SEQ ID NO: 184) FNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 112  Lip-S5D1-S6D4_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 5 with disulfide bond type 1 and OspA serotype 6 with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTG AAGAATGCTCTGAAASEQ ID NO: 113  Lip-S5D1-S6D4_His_aa Heterodimer fusion protein of OspA serotype 5 with disulfide bond type 1 and OspA serotype 6 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation, C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGLEHHHHHH SEQ ID NO: 114 Lip-S5D1-S6D4_His_nt  Coding sequence for heterodimer fusion protein of OspA serotype 5 with disulfide bond type 1 and OspA serotype 6 with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag  (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 115  S2D4-S1D1_aa Heterodimer fusion protein of OspA serotype 2 with disulfide bond type 4 and OspA serotype 1 with disulfide bond type 1, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) FNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALK SEQ ID NO: 116  Lip-S2D4-S1D1_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 2 with disulfide bond type 4 and OspA serotype 1 with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184),aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGT AATGCACTGAAASEQ ID NO: 117  Lip-S2D4-S1D1_His_aa Heterodimer fusion protein of OspA serotype 2 with disulfide bond type 4 and OspA serotype 1 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), N-terminal  lipidation, C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGLEHHHHHH SEQ ID NO: 118 Lip-S2D4-S1D1_His_nt  Coding sequence for heterodimer fusion protein of OspA serotype 2 with disulfide bond type 4 and OspA serotype 1 with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAATGCGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAAGAAGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAACAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAATGTACGATCACCGTGCAGAAATACGATAGTGCGGGTACCAACCTGGAAGGCACCGCTGTTGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGGCACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAATGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAAACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTGGAAATCACGAAACTGGATGAAATCTGTAATGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 119  S2D1-S1D4_aa Heterodimer fusion protein of OspA serotype 2 with disulfide bond type 1 and OspA serotype 1 with disulfide bond type 4, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) FNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALK SEQ ID NO: 120  Lip-S2D1-S1D4_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 2 with disulfide bond type 1 and OspA serotype 1 with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184),aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAA AATGCACTGAAASEQ ID NO: 121  Lip-S2D1-S1D4_His_aa Heterodimer fusion protein of OspA serotype 2 with disulfide bond type 1 and OspA serotype 1 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), N-terminal  lipidation, C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGLEHHHHHH SEQ ID NO: 122 Lip-S2D1-S1D4_His_nt  Coding sequence for heterodimer fusion protein of OspA serotype 2 with disulfide bond type 1 and OspA serotype 1 with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGAAAAAGGCGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTGTGTAACGCCCTGAAAGGCACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAGTCTCGGAAAAAATCATTACCCGTGCTTGCGGCACCCGTCTGGAATACACCGGCATTAAATCGGATGGCAGCGGCAAAGCGAAGGAAGTTCTGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAGACCACCCTGGTGGTGAAAGAAGGCACCGTTACGCTGAGCAAAAACATTAGTAAGTCCGGTGAAGTCTCTGTGGAACTGAATGATACCGACAGCTCTGCGGCCACCAAAAAGACGGCAGCTTGGAACTCAGGCACCTCGACGCTGACCATTACGGTTAATTCCAAAAAGACCAAAGATCTGGTCTTCACGAAAGAATGCACCATCACGGTGCAGCAATATGACAGCAACGGTACCAAACTGGAAGGCTCTGCGGTGGAAATCACGAAACTGGATGAAATCAAAAATGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 123  S4D4-S3D1_aa Heterodimer fusion protein of OspA serotype 4 with disulfide bond type 4 and OspA serotype 3 with disulfide bond type 1, LN1 peptide linker  (SEQ ID NO: 184) FNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK SEQ ID NO: 124  Lip-S4D4-S3D1_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 4 with disulfide bond type 4 and OspA serotype 3 with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTG TGTGCGGCACTGAAASEQ ID NO: 125  Lip-S4D4-S3D1_His_aa Heterodimer fusion protein of OspA serotype 4 with disulfide bond type 4 and OspA serotype 3 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal CSS for addition of lipids, N-terminal lipidation, C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGLEHHHHHH SEQ ID NO: 126 Lip-S4D4-S3D1_His_nt  Coding sequence for heterodimer fusion protein of OspA serotype 4 with disulfide bond type 4 and OspA serotype 3 with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag  (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGCTAAAGGTGAACTGTCGGAAAAAACCATCCTGCGCGCCTGTGGCACCCGCCTGGAATACACGGAAATCAAGTCGGACGGCACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCGACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAATGCACGATCACCGTTCAAAAATATGATTCCGCAGGTACCAACCTGGAAGGCAACGCTGTGGAAATCAAAACCCTGGACGAACTGAAAAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGTGTGCGGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 127  S4D1-S3D4_aa Heterodimer fusion protein of OspA serotype 4 with disulfide bond type 1 and OspA serotype 3 with disulfide bond type 4, LN1 peptide linker  (SEQ ID NO: 184) FNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALK SEQ ID NO: 128  Lip-S4D1-S3D4_nt Heterodimer fusion protein of OspA serotype 4 with disulfide bond type 1 and OspA serotype 3 with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTG AAGGCGGCACTGAAASEQ ID NO: 129  Lip-S4D1-S3D4_His_aa Heterodimer fusion protein of OspA serotype 4 with disulfide bond type 1 and OspA serotype 3 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation, C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGLEHHHHHH SEQ ID NO: 130 Lip-S4D1-S3D4_His_nt  Coding sequence for heterodimer fusion protein of OspA serotype 4 with disulfide bond type 1 and OspA serotype 3 with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag  (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGGCACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGAACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCCACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAACTGTCAGAAAAAGTGGTCACCCGCGCTTGTGGCACCCGCCTGGAATACACCGAAATCAAAAACGACGGCTCGGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGGAAGGCACCGTTACGCTGTCTAAAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAATGCACGATCACCGTGCAGAACTATAATCGTGCCGGTAATGCTCTGGAAGGCTCCCCGGCTGAAATCAAGGACCTGGCGGAACTGAAGGCGGCACTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 131  S6D4-S5D1_aa Heterodimer fusion protein of OspA serotype 6 with disulfide bond type 4 and OspA serotype 5 with disulfide bond type 1, LN1 peptide linker  (SEQ ID NO: 184) FNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 132  Lip-S6D4-S5D1_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 6 with disulfide bond type 4 and OspA serotype 5 with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTG TGTAATGCTCTGAAASEQ ID NO: 133  Lip-S6D4-S5D1_His_aa Heterodimer fusion protein of OspA serotype 6 with disulfide bond type 4 and OspA serotype 5 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation, C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGLEHHHHHH SEQ ID NO: 134 Lip-S6D4-S5D1_His_nt  Coding sequence for heterodimer fusion protein of OspA serotype 6 with disulfide bond type 4 and OspA serotype 5 with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag  (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGTGAAAAAACGATTGTTCGCGCCTGTGGCACCCGCCTGGAATACACGGATATCAAGTCGGATGGTTCGGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGGAAGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGACAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAATGCACGATCACCGTTCAACGCTATGATAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGGCACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAGATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 135  S6D1-S5D4_aa Heterodimer fusion protein of OspA serotype 6 with disulfide bond type 1 and OspA serotype 5 with disulfide bond type 4, LN1 peptide linker  (SEQ ID NO: 184) FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 136  Lip-S6D1-S5D4_nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 6 with disulfide bond type 1 and OspA serotype 5 with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTG AAGAATGCTCTGAAASEQ ID NO: 137  Lip-S6D1-S5D4_His_aa Heterodimer fusion protein of OspA serotype 6 with disulfide bond type 1 and OspA serotype 5 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation, C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) LipCSSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGLEHHHHHH SEQ ID NO: 138 Lip-S6D1-S5D4_His_nt  Coding sequence for heterodimer fusion protein of OspA serotype 6 with disulfide bond type 1 and OspA serotype 5 with disulfide bond type 4, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), C-terminal His tag  (GLEHHHHHH) (SEQ ID NO: 214) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGTTGCTCAAGCTTCAACGGCAAAGGTGAAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATTAAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAAAAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGTGCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAACTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAAATCAGTGAAAAAACCATTGTGCGTGCGTGTGGCACCCGTCTGGAATATACCGACATCAAGAGCGATAAAACGGGTAAAGCGAAGGAAGTTCTGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACCGAAGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCCCTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGACTCAGGCACCTCGACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAATGCACGATCACCGTGCAAAACTATGATAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTGGAAATTACCACGCTGAAAGAACTGAAGAATGCTCTGAAAGGTCTCGAGCACCACCACCACCACCAC SEQ ID NO: 139 N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy)  propyl cysteine Pam₃C SEQ ID NO: 140  Lip-S2D0-His amino acids of positions 131-273 of Borrelia afzelii strain K78, OspA serotype 2, wild-type sequence, N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNALKGLEHHHHHHSEQ ID NO: 141  Lip-S2D1-His aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 1 (aa 182 and 269), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKV ANDKVTLEVK CGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE L C NALKGLEHHHHHHSEQ ID NO: 142  Lip-S2D2-His aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 2 (aa 182 and 272), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKV ANDKVTLEVK CGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNA C KGLEHHHHHHSEQ ID NO: 143  Lip-S2D3-His aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 3 (aa 244 and 259), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTI C VQKYDSAGTNLEGT C VEIKTLDE LKNALKGLEHHHHHHSEQ ID NO: 144  Lip-S2D4-His aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 4 (aa 141 and 241), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCKQNELSAKTMTRE C GTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSK TSTLTISVNSKKTTQLVFTKQ CTITVQKYDSAGTNLEGTAVEIKTLDE LKNALKGLEHHHHHH SEQ ID NO: 145  Lip-S2D5-His aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 5 (aa 165 and 265), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKN C TLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKT C DE LKNALKGLEHHHHHHSEQ ID NO: 146  Lip-S2D6-His aa 131-273 of Borrelia afzelii strain K78, OspA sero type 2 with disulfide bond type 6 (aa 185 and 272), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKV ANDKVTLEVKEGT CTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNA C KGLEHHHHHHSEQ ID NO: 147  Lip-S2D7-His aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 7 (aa 199 and 223), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVT C ALNDTNTTQATKKTGAWDSK TST CTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNALKGLEHHHHHHSEQ ID NO: 148  Lip-S2D8-His aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 8 (aa 243 and 262), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSK TSTLTISVNSKKTTQLVFTKQDTC TVQKYDSAGTNLEGTAVE C KTLDE LKNALKGLEHHHHHH SEQ ID NO: 149 Lip-S2D9-His  aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 9 (aa 184 and 204), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKV ANDKVTLEVKEG CVTLSKEIAKSGEVTVALND C NTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNALKGLEHHHHHHSEQ ID NO: 150  Lip-S2D10-His aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 10 (aa 201 and 214), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVA C NDTNTTQATKKT C AWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNALKGLEHHHHHHSEQ ID NO: 151  Lip-S2D11-His aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 11 (aa 246 and 259), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITV C KYDSAGTNLEGT C VEIKTLDE LKNALKGLEHHHHHHSEQ ID NO: 152  Lip-S2D12-His aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 12 (aa 167 and 178), N-terminal CKQN for addition of lipids, C-terminal His tag (GLEHHHHHH)  (SEQ ID NO: 214) LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFT C EGKV ANDKVT CEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNALKGLEHHHHHHSEQ ID NO: 153  Lip-S2D0  amino acids of positions 131-273 of Borrelia afzelii strain K78, OspA serotype 2, wild-type sequence, N-terminal CKQN for addition of lipids LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNALK SEQ ID NO: 154 Lip-S2D1  aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 1 (aa 182 and 269), N-terminal CKQN for addition of lipids LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKV ANDKVTLEVK CGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE L C NALKSEQ ID NO: 155  Lip-S2D2 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 2 (aa 182 and 272), N-terminal CKQN for addition of lipids LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKV ANDKVTLEVK CGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNA C KSEQ ID NO: 156  Lip-S2D3 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 3 (aa 244 and 259), N-terminal CKQN for addition of lipids LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTI C VQKYDSAGTNLEGT C VEIKTLDE LKNALKSEQ ID NO: 157  Lip-S2D4 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 4 (aa 141 and 241), N-terminal CKQN for addition of lipids  LipCKQNELSAKTMTRE CGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSK TSTLTISVNSKKTTQLVFTKQ CTITVQKYDSAGTNLEGTAVEIKTLDE LKNALK SEQ ID NO: 158  Lip-S2D5 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 5 (aa 165 and 265), N-terminal CKQN for addition of lipids LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKN C TLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKT C DE LKNALKSEQ ID NO: 159  Lip-S2D6 aa 131-273 of Borrelia afzelii strain K78, OspA sero type 2 with disulfide bond type 6 (aa 185 and 272), N-terminal CKQN for addition of lipids LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKV ANDKVTLEVKEGT CTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNA C KSEQ ID NO: 160  Lip-S2D7 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 7 (aa 199 and 223), N-terminal CKQN for addition of lipids LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVT C ALNDTNTTQATKKTGAWDSK TST CTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNALK SEQ ID NO: 161 Lip-S2D8  aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 8 (aa 243 and 262), N-terminal CKQN for addition of lipids LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSK TSTLTISVNSKKTTQLVFTKQDTC TVQKYDSAGTNLEGTAVE C KTLDE LKNALK SEQ ID NO: 162  Lip-S2D9 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 9 (aa 184 and 204), N-terminal CKQN for addition of lipids LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKV ANDKVTLEVKEG CVTLSKEIAKSGEVTVALND C NTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNALK SEQ ID NO: 163 Lip-S2D10  aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 10 (aa 201 and 214), N-terminal CKQN for addition of lipids LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVA C NDTNTTQATKKT C AWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNALK SEQ ID NO: 164 Lip-S2D11  aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 11 (aa 246 and 259), N-terminal CKQN for addition of lipids LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITV C KYDSAGTNLEGT C VEIKTLDE LKNALKSEQ ID NO: 165  Lip-S2D12 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 12 (aa 167 and 178), N-terminal CKQN for addition of lipids LipCKQNELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFT C EGKV ANDKVT CEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDE LKNALK SEQ ID NO: 166 S2D0  amino acids of positions 131-273 of Borrelia afzelii strain K78, OspA serotype 2, wild-type  sequence ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 167  S2D1 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 1 (aa 182  and 269) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTL EVK CGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDEL C NALK SEQ ID NO: 168  S2D2 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 2 (aa 182  and 272) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTL EVK CGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNA C K SEQ ID NO: 169  S2D3 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 3 (aa 244  and 259) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTIS VNSKKTTQLVFTKQDTI CVQKYDSAGTNLEGT C VEIKTLDELKNALK SEQ ID NO: 170  S2D4 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 4 (aa 141  and 241)  ELSAKTMTRE CGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTIS VNSKKTTQLVFTKQ CTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 171  S2D5 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 5 (aa 165  and 265) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKN C TLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKT C DELKNALK SEQ ID NO: 172  S2D6 aa 131-273 of Borrelia afzelii strain K78, OspA sero type 2 with disulfide bond type 6 (aa  185 and 272) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTL EVKEGT CTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNA C K SEQ ID NO: 173  S2D7 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 7 (aa 199  and 223) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTL EVKEGTVTLSKEIAKSGEVT CALNDTNTTQATKKTGAWDSKTST C TISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 174  S2D8 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 8 (aa 243  and 262) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTIS VNSKKTTQLVFTKQDT CTVQKYDSAGTNLEGTAVE C KTLDELKNALK SEQ ID NO: 175  S2D9 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 9 (aa 184  and 204) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTL EVKEG CVTLSKEIAKSGEVTVALND C NTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 176  S2D10 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 10 (aa  201 and 214) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTL EVKEGTVTLSKEIAKSGEVTVAC NDTNTTQATKKT C AWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 177  S2D11 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 11 (aa  246 and 259) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTIS VNSKKTTQLVFTKQDTITV CKYDSAGTNLEGT C VEIKTLDELKNALK SEQ ID NO: 178  S2D12 aa 131-273 of Borrelia afzelii strain K78, OspA serotype 2 with disulfide bond type 12 (aa  167 and 178) ELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFT C EGKVANDKVT CEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 179 B. burgdorferi s.s. (strain B31, serotype 1), OspA_aa 126-273 with replaced hLFA-like  sequence from serotype 1 OspA FNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIK NALK SEQ ID NO: 180 B. garinii (strain PBr, serotype 3), OspA_aa  126-274 FNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAEL KAALK SEQ ID NO: 181 B. bavariensis (strain PBi, serotype 4), OspA_aa  126-273 FNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELK NALK SEQ ID NO: 182 B. garinii (strain PHei, serotype 5), OspA_aa  126-273 FNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELK NALK SEQ ID NO: 183 B. garinii (strain DK29, serotype 6), OspA_aa  126-274 FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKEL KNALK SEQ ID NO: 184 LN1 peptide linker constructed from two separate loop regions of the N-terminal half of OspA from B. burgdorferi s.s. strain B31 (aa 65-74 and aa 42-53, amino acid exchange at  position 53: D53S) GTSDKNNGSGSKEKNKDGKYS  SEQ ID NO: 185  Lip-S1D4-S2D4_aa Heterodimer fusion protein of OspA serotypes 1 and 2 both with disulfide bond type 4, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18) LipCSSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 186  Lip-S1D1-S2D1_aa Heterodimer fusion protein of OspA serotype 1 and OspA serotype 2 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18),  N-terminal lipidation LipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK SEQ ID NO: 187  Lip-S3D4-S4D4_aa Heterodimer fusion protein of OspA serotype 3 and OspA serotype 4 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184),  N-terminal lipidation LipCSSFNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALK SEQ ID NO: 188  Lip-S3D1-S4D1_aa Heterodimer fusion protein of OspA serotypes 3 and 4 both with disulfide bond type 1, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALK SEQ ID NO: 189  Lip-S5D4-S6D4_aa Heterodimer fusion protein OspA serotypes 5 and 6 both with disulfide bond type 4, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 190  Lip-S5D1-S6D1_aa Heterodimer fusion protein of OspA serotypes 6 both with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 191  Lip-S2D4-S1D4_aa Heterodimer fusion protein of OspA serotypes 2 and 1 both with disulfide bond type 4, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18),  N-terminal lipidation LipCSSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALK SEQ ID NO: 192  Lip-S2D1-S1D1_aa Heterodimer fusion protein of OspA serotypes 2 and 1 both with disulfide bond type 1, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18),  N-terminal lipidation LipCSSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALK SEQ ID NO: 193  Lip-S4D4-S3D4_aa Heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfide bond type 4, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALK SEQ ID NO: 194  Lip-S4D1-S3D1_aa Heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfide bond type 1, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK SEQ ID NO: 195  Lip-S6D4-S5D4_aa Heterodimer fusion protein of OspA serotypes 6 and 5 both with disulfide bond type 4, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 196  Lip-S6D1-S5D1_aa Heterodimer fusion protein of OspA serotypes 6 and 5 both with disulfide bond type 1, N- terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 197  Lip-S1D4-S2D1_aa Heterodimer fusion protein of OspA serotype 1 with disulfide bond type 4 and OspA serotype 2 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), N-terminal lipidation LipCSSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK SEQ ID NO: 198  Lip-S1D1-S2D4_aa Heterodimer fusion protein of OspA serotype 1 with disulfide bond type 1 and OspA serotype 2 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), N-terminal lipidation LipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 199  Lip-S3D4-S4D1_aa Heterodimer fusion protein of OspA serotype 3 with disulfide bond type 4 and OspA serotype 4 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNEKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALK SEQ ID NO: 200  Lip-S3D1-S4D4_aa Heterodimer fusion protein of OspA serotype 3 with disulfide bond type 1 and OspA serotype 4 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNEKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALKGTSDKNNGSGSKEKNKDGKYSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALK SEQ ID NO: 201  Lip-S5D4-S6D1_aa Heterodimer fusion protein of OspA serotype 5 with disulfide bond type 4 and OspA serotype 6 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 202  Lip-S5D1-S6D4_aa Heterodimer fusion protein of OspA serotype 5 with disulfide bond type 1 and OspA serotype 6 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 203  Lip-S2D4-S1D1_aa Heterodimer fusion protein of OspA serotype 2 with disulfide bond type 4 and OspA serotype 1 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), N-terminal lipidation LipCSSFNEKGELSAKTMTRECGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQCTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALK SEQ ID NO: 204  Lip-S2D1-S1D4_aa Heterodimer fusion protein of OspA serotype 2 with disulfide bond type 1 and OspA serotype 1 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO: 18), N-terminal lipidation LipCSSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKIITRACGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKECTITVQQYDSNGTKLEGSAVEITKLDEIKNALK SEQ ID NO: 205  Lip-S4D4-S3D1_aa Heterodimer fusion protein of OspA serotype 4 with disulfide bond type 4 and OspA serotype 3 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNAKGELSEKTILRACGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKECTITVQKYDSAGTNLEGNAVEIKTLDELKNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK SEQ ID NO: 206  Lip-S4D1-S3D4_aa Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 4 with disulfide bond type 1 and OspA serotype 3 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTRACGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKECTITVQNYNRAGNALEGSPAEIKDLAELKAALK SEQ ID NO: 207  Lip-S6D4-S5D1_aa Heterodimer fusion protein of OspA serotype 6 with disulfide bond type 4 and OspA serotype 5 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNGKGETSEKTIVRACGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKECTITVQRYDSAGTNLEGKAVEITTLKELKNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 208  Lip-S6D1-S5D4_aa Heterodimer fusion protein of OspA serotype 6 with disulfide bond type 1 and OspA serotype 5 with disulfide bond type 4, N-terminal CSS for addition of lipids, LN1 peptide linker (SEQ ID NO: 184), N-terminal lipidation LipCSSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEISEKTIVRACGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKECTITVQNYDSAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 209 B. afzelii (strain K78; OspA serotype 2) aa 17-273, lipidation signal sequence removed (aa 1-16: MKKYLLGIGLILALIA, (SEQ ID NO: 14)), C-terminal His tag (GLEHHHHHH) (SEQ ID NO: 214) CKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIELKGTSDKDNGSGVLEGTKDDKSKAKLTIADDLSKTTFELFKEDGKTLVSRKVSSKDKTSTDEMFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELKNALKGLEHHHHHH SEQ ID NO: 210 Cysteine-containing peptide from E. coli  CSS  SEQ ID NO: 211 Cysteine-containing peptide from OspA  CKQN  SEQ ID NO: 212 amino acids of positions 126-274 of B. garinii strain T25, OspA serotype 7 FNDKGKLSEKVVTRANGTRLEYTEIQNDGSGKAKEVLKSLTLEGTLTADGETKLTVEAGTVTLSKNISESGEITVELKDTETTPADKKSGTWDSKTSTLTISKNSQKTKQLVFTKENTITVQKYNTAGTKLEGSPAEIKDLEAL KAALK SEQ ID NO: 213 Forward oligonucleotide primer for the recA gene of all relevant Borrelia species causing Lyme  borreliosis CATGCTCTTGATCCTGTTTA  SEQ ID NO: 214  C-terminal His tag  GLEHHHHHH SEQ ID NO: 215  Reverse oligonucleotide primer for the recA gene of all relevant Borrelia species causing  Lyme borreliosis CCCATTTCTCCATCTATCTC 

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference.

What is claimed is: 1.-19. (canceled)
 20. A method for producing apolypeptide comprising a mutant fragment of an outer surface protein A(OspA), wherein the mutant OspA fragment is defined by SEQ ID NO: 216,or a functional variant thereof, the method comprising the followingsteps: a) introducing a vector encoding the polypeptide into a hostcell, b) growing the host cell under conditions allowing for expressionof said polypeptide, c) homogenizing said host cell, and d) subjectingthe host cell homogenate to purification steps.
 21. The method accordingto claim 20, wherein said functional variant has at least 95% sequenceidentity to the wild-type OspA fragment.
 22. The method according toclaim 20, wherein the polypeptide comprises a heterodimer selected fromthe group consisting of Lip-S1D1-S2D1 (SEQ ID NO: 186), Lip-S2D1-S1D1(SEQ ID NO: 192), Lip-S1D1-S2D4 (SEQ ID NO: 198) and Lip-S2D4-S1D1 (SEQID NO: 203).
 23. The method according to claim 20, wherein thepolypeptide consists of a heterodimer selected from the group consistingof Lip-S1D1-S2D1 (SEQ ID NO: 186), Lip-S2D1-S1D1 (SEQ ID NO: 192),Lip-S1D1-S2D4 (SEQ ID NO: 198) and Lip-S2D4-S1D1 (SEQ ID NO: 203). 24.The method according to claim 20, wherein the vector comprises a nucleicacid molecule encoding said polypeptide.
 25. The method according toclaim 24, wherein said nucleic acid molecule encoding said polypeptideis defined by SEQ ID NO:
 48. 26. The method according to claim 20,wherein said vector is pET28b(+).
 27. The method according to claim 20,wherein said host cell is E. coli.
 28. The method according to claim 27,wherein said E. coli is an E. coli BL21 or an E. coli BL21 Star™ cell.29. The method according to claim 20, wherein said purification stepscomprise enriching the polypeptide in the lipid phase by phaseseparation and purifying over a gel filtration column.
 30. The methodaccording to claim 29, wherein said purification steps further compriseprocessing over a buffer exchange column.
 31. A method for producing apharmaceutical composition comprising a polypeptide comprising a mutantfragment of an outer surface protein A (OspA), wherein the mutant OspAfragment is defined by SEQ ID NO: 216, or a functional variant thereof,the method comprising combining said polypeptide with one or morepharmaceutically acceptable carriers or excipients.
 32. The methodaccording to claim 31, wherein said functional variant has at least 95%sequence identity to the wild-type OspA fragment.
 33. The methodaccording to claim 31, wherein said polypeptide comprises a heterodimerselected from the group consisting of Lip-S1D1-S2D1 (SEQ ID NO: 186),Lip-S2D1-S1D1 (SEQ ID NO: 192), Lip-S1D1-S2D4 (SEQ ID NO: 198) andLip-S2D4-S1D1 (SEQ ID NO: 203).
 34. The method according to claim 31,wherein said polypeptide consists of a heterodimer selected from thegroup consisting of Lip-S1D1-S2D1 (SEQ ID NO: 186), Lip-S2D1-S1D1 (SEQID NO: 192), Lip-S1D1-S2D4 (SEQ ID NO: 198) and Lip-S2D4-S1D1 (SEQ IDNO: 203).
 35. The method according to claim 31, wherein saidpharmaceutical composition comprises Lip-S1D1-S2D1 (SEQ ID NO: 186) andLip-S5D1-S6D1 (SEQ ID NO: 190).
 36. The method according to claim 31,wherein said one or more pharmaceutically acceptable carriers orexcipients are selected from the group comprising saline, bufferedsaline, dextrose, water, glycerol, ethanol and adjuvants.
 37. The methodaccording to claim 36, wherein said buffered saline is phosphatebuffered saline.
 38. The method according to claim 36, wherein saidadjuvant is aluminium hydroxide.
 39. The method according to claim 31,wherein said pharmaceutical composition is a vaccine.